General amyloid interaction motif (gaim)

ABSTRACT

The present invention relates to variants of the general amyloid interaction motif (GAIM) of bacteriophage gene 3 protein (g3p) and fusion proteins thereof. The GAIM variants and fusion proteins of the invention are partially or fully deimmunized and demonstrate superior binding and specificity to a diverse array of amyloid proteins, and exhibit enhanced amyloid remodeling and inhibition of amyloid aggregation. The present invention further relates to nucleic acids, vectors, host cells, and methods of making the GAIM variants and fusion proteins thereof. The present invention also relates to pharmaceutical compositions and methods of increasing bacteriophage infectivity, methods of detecting amyloid aggregates, and methods of diagnosing and/or treating a disease associated with misfolded and/or aggregated amyloid protein.

This application claims priority to U.S. Provisional Application No.62/685,757, filed Jun. 15, 2018, and U.S. Provisional Application No.62/749,499, filed Oct. 23, 2018, the disclosure of which is incorporatedherein by reference in their entirety.

The invention relates to polypeptides comprising a variant of thegeneral amyloid interaction motif (GAIM) of filamentous bacteriophagegene 3 protein (“g3p,” also known as “p3” or “pill”) such that thepolypeptides are partially or fully deimmunized and demonstrate superiorpotency, superior structural stability, and increased bindingspecificity to amyloid proteins relative to the prior art. Nucleic acidmolecules and constructs encoding such polypeptides, host cellstransformed with such nucleic acid molecules, and methods of making suchpolypeptides recombinantly are encompassed. In addition, the inventionrelates to diagnostic and pharmaceutical compositions comprising thepolypeptides disclosed herein, to the use of diagnostic compositions todetect amyloid aggregates and/or to diagnose a disease associated withmisfolded and/or aggregated protein, and to the therapeutic and/orprophylactic use of pharmaceutical compositions to decrease amyloidload, prevent aggregation, disaggregate amyloid, or otherwise treat orprevent a disease associated with misfolded and/or aggregated amyloidprotein, such as systemic and peripheral amyloid diseases,neurodegenerative diseases including neurodegenerative tauopathies, andtransmissible spongiform encephalopathies (prion-associated diseases).Polypeptides of the invention include fusion proteins andamyloid-binding portions thereof.

Bacteriophage g3p directly binds amyloid fibers, andbacteriophage-mediated amyloid disaggregation (e.g., remodeling) isdependent upon this initial binding step. See, e.g., WO 2013/082114 A1,hereby incorporated by reference in its entirety. The inventorspreviously identified a minimal sequence of g3p required for amyloidbinding, amyloid disaggregation, and/or prevention of formation ofamyloid aggregates. Id. This minimal sequence is encompassed by thegeneral amyloid interaction motif (GAIM), which comprises the N1 and N2domains of g3p, and led to the generation of g3p polypeptides (includingmutants, fragments, fusion proteins, or pharmaceutical compositionsthereof) that are capable of binding to amyloid protein and/ordisaggregating amyloid proteins. See id.; WO 2014/055515 A1, herebyincorporated by reference in its entirety. The g3p polypeptidesdisclosed in WO 2013/082114 A1 and WO 2014/055515 A1 are effective forthe prevention or treatment of diseases associated with misfolded and/oraggregated amyloid proteins. Id. However, these g3p polypeptides alsocomprise human T-cell epitopes that may elicit an unwanted immuneresponse in patients. Partially deimmunized g3p polypeptides weretherefore developed, comprising mutations that remove up to four of thefive T-cell epitopes present in native g3p. See WO 2014/193935 A1,hereby incorporated by reference in its entirety.

Further, the recombinant production in animal cells of g3p polypeptidesthat bind amyloid—and are useful for detection, diagnosis, prevention,or treatment of amyloid-related diseases or conditions—was limited bythe presence of a potential N-glycosylation site. Thus, improved g3ppolypeptides, including partially deimmunized g3p polypeptides, weregenerated in which the potential N-glycosylation site was removed by oneor more mutations. See WO 2016/090022 A8, hereby incorporated byreference in its entirety.

Despite these advances, there remains a need in the art for g3ppolypeptides that are more stable, potent, and specific as well as fullyor almost fully deimmunized.

Generating polypeptides with these sets of therapeutic qualities has sofar posed a significant challenge, at least in part due to certaininverse relationships in the g3p polypeptides: First, our prior attemptsto remove T-cell epitope 2, which is required for full deimmunization ofa g3p polypeptide, have consistently resulted in reduced amyloid-bindingability. Second, our prior attempts to increase the stability of theN1-N2 domains of GAIM have also consistently caused reducedamyloid-binding activity. For example, a super-stabilized GAIM variant,PB113, exhibits poor amyloid-binding capability. Third, instability ofthe N2 domain drives promiscuous interactions with non-amyloidsubstrates like soluble proteins, sticky molten-globule structures andnon-amyloid fibrillar polymers, such as collagen and elastin. Thus,destabilizing N2 to increase amyloid-binding activity sacrificesspecificity, while stabilizing N2 to avoid off-target binding sacrificesbinding to amyloid. Surprisingly, all three inverse relationships wereovercome by generating fusion proteins comprising GAIM variants andfusions thereof in an “open-stabilized” conformation.

Unlike the prior art, the open-stabilized polypeptides described hereindemonstrate potent amyloid-binding and remodeling activities across awide array of amyloids while maintaining protein stability and bindingspecificity. Thus, in some aspects, the present invention relates toopen-stabilized variants of GAIM (GAIM), GAIM-immunoglobulin (GAIM-Ig)fusion proteins, or pharmaceutical compositions thereof, that are atleast partially deimmunized and have superior amyloid-binding activity,amyloid-binding specificity, and amyloid remodeling activity. In someaspects, these open-stabilized GAIM variants, GAIM-Ig fusion proteins,or pharmaceutical compositions thereof are fully deimmunized withoutsacrificing potent and specific amyloid binding and without sacrificingstructural stability. The open-stabilized GAIM variants and GAIM-Igfusions described herein are capable of engaging and removing amyloidsin brain and peripheral organs and constitute a novel set ofpolypeptides with superior ability to detect, diagnose, prevent, delayonset of and/or treat diseases associated with misfolded and/oraggregated amyloid protein.

Additional objects and advantages of the invention are set forth in partin the description which follows, and will be obvious from thedescription, or may be learned by practice of the invention. The objectsand advantages of the invention will be realized and attained by meansof the elements and combinations particularly pointed out in theappended claims. It is to be understood that both the foregoing generaldescription and the following detailed description are exemplary andexplanatory only and are not restrictive of the invention as claimed.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a graphical representation of the tertiary structure of theGAIM N1 and N2 domains (shown using PDB structure 2G3P). β-strandssubjected to site-directed mutagenesis are shown in dark grey. Arrowsindicate the location of polar residues in N1 and hydrophobic residuesin N2. FIG. 1B is a graphical representation of a GAIM-Ig fusion of theinvention. FIG. 1C is a graphical representation of a GAIM dimer.

FIG. 2 depicts results of hydrogen/deuterium (H/D) exchange studiesshowing the GAIM-interacting sequences in fAβ42.

FIG. 3A compares corresponding portions of the amino acid sequence ofthe N1 portion of g3p from fd phage (SEQ ID NO:1) and the correspondingN1 portion of g3p from IF1 phage (SEQ ID NO:2). The open box depicts, ontop, the amino acids 23-28 (DDKTLD; SEQ ID NO:3) of PB106 and the GAIMscaffold PB120 (derived from PB106) and, on bottom, the EGDS (SEQ ID NO:4) substitution present in the open-stabilized GAIM-Ig fusions of theinvention (EGDS=SEQ ID NO:4). FIG. 3B is a graphical representation ofthe tertiary structure of the GAIM N1 and N2 domains (shown using PDBstructure 2G3P). The open box in FIG. 3B shows the location of SEQ IDNO:3 in fd g3p.

FIG. 3C is a graphical representation of three slow-folding loops (alsoreferred to as “turns”) implicated in stabilization of the N2 domain.T1=Turn 1 (FQNN; SEQ ID NO: 5); T2=Turn 2 (RQGA; SEQ ID NO: 6); T3=Turn3 (QGTDPVK; SEQ ID NO: 7). As demonstrated below, removing one or moreof T1, T2, or T3 by mutagenesis stabilizes the N2 domain. FIG. 3Ddepicts changes in binding and stability based on select amino acidsubstitutions of T50.

FIGS. 4A and 4B show representative data from thermal meltingexperiments of a GAIM-Ig fusion compared to a GAIM dimer or monomer.Thermal melting was monitored by SYPRO® Orange binding. The firsttransition, Tm1, was calculated by non-linear fitting from normalizedfluorescence intensities.

FIGS. 5A and 5B depict fluorescence emission spectra of GAIM at 0 Mguanidine hydrochloride (GuHCI) (broken lines), 2 M GuHCI (continuouslines), and 5 M GuHCI (dotted lines) excited at 295 nm (FIG. 5A) and 280nm

(FIG. 5B). FIG. 5C depicts equilibrium unfolding of GAIM dimer by GuHCI.FIG. 5C shows two transitions, the first between 1 M and 2 M GuHCI andthe second between 2 M and 4 M GuHCI. Relative fluorescence intensitiesat 340 nm (excitation 280 nm) were plotted at various concentrations ofGuHCI. FIG. 5D depicts N2 domain unfolding at 1.5 M GuHCI. FIG. 5Edepicts N1 domain unfolding at 2.6 M GuHCI.

FIGS. 6A and 6B depict the mapping of GAIM residues that modulateamyloid binding. FIG. 6A is a scatter plot showing the Aβ42fiber-binding potency of GAIM variants correlating with Tm1 (Spearmancorrelation coefficient=0.703, p<0.0001). FIG. 6B is a scatter plotshowing the ftau fiber-binding potency of GAIM variants correlating withTm1 (Spearman correlation coefficient=0.878, p<0.0001). Variants withpoor ftau binding are presented EC₅₀=1000 nM due to inaccuracy in thecurve fit for variants that does not reach saturation in the ELISA. Forboth FIG. 6A and FIG. 6B, the GAIM scaffold PB120 is represented by agrey triangle and variants are represented by circles. A decrease in Tm1indicates a more open conformation of GAIM, resulting in increasedbinding, whereas stabilized variants with higher Tm1 tend to losebinding activity.

FIGS. 7A and 7B show amyloid binding for select GAIM-Ig fusion proteins.FIG. 7A compares amyloid binding for the GAIM scaffold (closed circle)and stabilized variants thereof. FQGN, VNGV, and QGGK are SEQ IDNOs:8-10, respectively. FIG. 7B shows the superior binding ofopen-stabilized (but not super-stabilized) GAIM-Ig fusion proteins.Open-stabilized polypeptides, (other than the super-stabilizedpolypeptide PB113, shown on the far right), are indicated within theopen box.

FIGS. 8A-8D shows binding of a representative open-stabilized GAIM-Igfusion (circles) to various Aβ fibers, compared to binding of thecontrol scaffold to those Aβ fibers (squares). FIG. 8A shows binding toAβ3-42-Pyro fibers; FIG. 8B shows binding to Aβ1-42 E22Q fibers; FIG. 8Cshows binding to Aβ11-42 fibers; FIG. 8D shows binding to Aβ11-42-Pyrofibers. The aggregates used for these experiments show very diversemorphology that range from long unbranched fibers (A61-42 E22Q fibers)to highly zig-zagged conformers (Aβ3-42-Pyro fibers).Pyro=pyro-glutamate.

FIG. 9 shows the effect of N2-stabilizing mutations on off-targetbinding to collagen. FQGN=SEQ ID NO:8, VNGV=SEQ ID NO:9; QGGK=SEQ IDNO:10.

FIGS. 10A-10D address the remodeling efficiencies of different GAIM-Igfusion proteins incubated with Aβ42 fibers. FIG. 10A further shows acomparison to remodeling effected by 6E10 MAb. Circles=mean;Bars=standard deviation. FIG. 10B further demonstrates the correlationbetween Aβ42 binding and remodeling by various GAIM-Ig fusion proteins.Open-stabilized GAIM-Ig-fusions are represented by dark grey upside-downtriangles. The GAIM scaffold is depicted by a light grey right-side-uptriangle. Circles represent other tested GAIM-Ig fusions. FIG. 10Ccompares the remodeling efficiency of a representative open-stabilizedpolypeptide (circles) against the remodeling efficiency of the GAIMscaffold (triangles) or fiber alone (squares). Bars=standard deviation.Greater remodeling efficiency is demonstrated by greater dissolution offibers in urea (e.g., lower ThT fluorescence). FIG. 10D showstransmission electron microscopy (TEM) images showing Aβ42 fibers before(left) and after (right) incubation with 0.8 μM PB108 at 37° C. for sixdays.

FIGS. 11A-11C address the remodeling efficiencies of different GAIM-Igfusion proteins incubated with tau fibers. FIG. 11A compares theremodeling efficiencies of representative open-stabilized GAIM-Ig fusionprotein PB108 and the super-stabilized fusion protein PB113. Remodelingis indicated by the presence of tauKL monomers and dimers (middle panel)in treated aggregates. FIG. 11B compares the remodeling efficiencies oftwo representative open-stabilized GAIM-Ig fusion proteins and the GAIMscaffold, at different concentrations of fusion protein. Error barsrepresent standard deviation from three independent experiments. FIG.11C shows TEM images showing tauKL fibers before (left) and after(right) incubation with 100 nM PB108 at 37° C. for three days.

FIGS. 12A-12D depict inhibition of amyloid assembly by GAIM-Ig fusionproteins of the invention. Error bars represent standard deviation fromthree or more independent experiments. FIG. 12A showsconcentration-dependent inhibition of Aβ42 fiber assembly. PB120(control scaffold) is represented by circles, PB108 by squares, andPB116 by triangles. FIG. 12B compares inhibition of Aβ42 fiber assemblyat 250 nM GAIM fusion. FIG. 12C shows concentration-dependent inhibitionof tauKL fiber assembly. PB120 (control scaffold) is represented bycircles, PB108 by squares, and PB116 by triangles. FIG. 12D comparesinhibition of tauKL fiber assembly at 250 nM GAIM fusion.

Brief Description of Sequences

SEQ ID NO: 1 = AETVESCLAKPHTENSFTNVWKDDKTLDRYAN SEQ ID NO: 2 =ATTDAECLSKPAFDGTLSNVWKEGDSRYAN SEQ ID NO: 3 = DDKTLD; SEQ ID NO: 4 =EGDS SEQ ID NO: 5 = FQNN; SEQ ID NO: 6 = RQGA; SEQ ID NO: 7 = QGTDPVK;SEQ ID NO: 8 = FQGN; SEQ ID NO: 9 = VNGV; SEQ ID NO: 10 = QGGKSEQ ID NO: 11 = MAETVESCLAKPHTENSFTNVWKDDKTLDRYANYEGCLWNAGGVVVCTGDETQCYGTWVPIGLAIPENEGGGSEGGGSEGGGSEGGGTKPPEYGDTPIPGYTYINPLDGTYPPGTEQNPANPNPSLEESQPLNTFMFQNNRFRNRQGALTVYTGTFTQGTDPVKTYYQYTPVSSRAMYDAYWNGKFRDCAFHSGFNEDPFVCEYQGQSSDLPQPPANAGGESGGGSGGGSEGGGSEGGGSEGGGSEGGGSGGGSGSGARSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNVVYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDK SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKSEQ ID NO: 12 = MAETVESCLAKPHTENSFTNVWKDDKTLDRYANYEGCLWNAGGVVVCTGDETQCYGTWVPIGLAIPENEGGGSEGGGSEGGGSEGGGTKPPEYGDTPIPGYTYINPLDGTYPPGTEQNPANPNPSLEESQPLNTFMFQNNRFRNRQGALTVYTGTFTQGTDPVKTYYQYTPVSSRAMYDAYWNGKFRDCAFHSGFNEDPFVCEYQGQSSDLPQPPANAGGESGGGSG GGSEGGGSEGGGSEGGGSEGGGSGGGSGSGSEQ ID NO: 13 = MAETVESCLAKPHTENSFTNVWKDDKTLDRYANYEGCLWNAGGVVVCTGDETQCYGHWVPIGLAIPENEGGGSEGGGSEGGGSEGGGTKPPEYGDTPIPGYTYINPLDGTYPPGTEQNPANPNPSLEESQPLNTFMFQNNRFRNRQGALTVYTGTFTQGTDPVKTYYQYTPVSSRAMYDAYWNGKFRDCAFHSGFNEDPFVCEYQGQSSDLPQPPANAGGESGGGSGGGSEGGGSEGGGSEGGGSEGGGSGGGSGSGARSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNVVYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDK SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKSEQ ID NO: 14 = MAETVESCLAKPHTENSFTNVWKDDKTLDRYANYEGCLWNAGGVVVCTGDETQCYGHWVPIGLAIPENEGGGSEGGGSEGGGSEGGGTKPPEYGDTPIPGYTYINPLDGTYPPGTEQNPANPNPSLEESQPLNTFMFQNNRFRNRQGALTVYTGTFTQGTDPVKTYYQYTPVSSRAMYDAYWNGKFRDCAFHSGFNEDPFVCEYQGQSSDLPQPPANAGGESGGGSG GGSEGGGSEGGGSEGGGSEGGGSGGGSGSGSEQ ID NO: 15 = MAETVESCLAKPHTENSFTNVWKEGDSRYANYEGCLWNAGGVVVCTGDETQCYGHWVPIGLAIPENEGGGSEGGGSEGGGSEGGGTKPPEYGDTPIPGYTYINPLDGTYPPGTEQNPANPNPSLEESQPLNTFMFQNNRFRNRQGALTVYTGTFTQGTDPVKTYYQYTPVSSRAMYDAYWNGKFRDCAFHSGFNEDPFVCEYQGQSSDLPQPPANAGGESGGGSGGGSEGGGSEGGGSEGGGSEGGGSGGGSGSGARSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNVVYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR WQQGNVFSCSVMHEALHNHYTQKSLSLSPGKSEQ ID NO: 16 = MAETVESCLAKPHTENSFTNVWKEGDSRYANYEGCLWNAGGVVVCTGDETQCYGHWVPIGLAIPENEGGGSEGGGSEGGGSEGGGTKPPEYGDTPIPGYTYINPLDGTYPPGTEQNPANPNPSLEESQPLNTFMFQNNRFRNRQGALTVYTGTFTQGTDPVKTYYQYTPVSSRAMYDAYWNGKFRDCAFHSGFNEDPFVCEYQGQSSDLPQPPANAGGESGGGSGGG SEGGGSEGGGSEGGGSEGGGSGGGSGSGSEQ ID NO: 17 = MAETVESSLAKPHIEGSFTNVWKDDKTLDVVYANYEGILWKATGVVVITGDETQVYAIWVPVGLAIPENEGGGSEGGGSEGGGSEGGGTKPPEYGDTPIPGYIYINPLDGTYPPGTEQNPANPNPSLEESHPLNTFMFQGNRFRNRQGALTVYTGTFTQGTDPVKTYYQYTPVSSRAMYDAYWNGKFRDVAFHSGFNEDPLVAEYQGQLSYLPQPPANAGGESGGGSGGGSEGGGSEGGGSEGGGSEGGGSGGGSGSGARSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNVVYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 18 =MAETVESSLAKPHIEGSFTNVWKDDKTLDVVYANYEGILWKATGVVVITGDETQVYAIWVPVGLAIPENEGGGSEGGGSEGGGSEGGGTKPPEYGDTPIPGYIYINPLDGTYPPGTEQNPANPNPSLEESHPLNTFMFQGNRFRNRQGALTVYTGTFTQGTDPVKTYYQYTPVSSRAMYDAYWNGKFRDVAFHSGFNEDPLVAEYQGQLSYLPQPPANAGGESGGGS GGGSEGGGSEGGGSEGGGSEGGGSGGGSGSGSEQ ID NO: 19 = MAETVESCLAKPHTENSFTNVWKEGDSRYANYEGCLWNAGGVVVCTGDETQCYGHWVPIGLAIPENEGGGSEGGGSEGGGSEGGGTKPPEYGDTPIPGYTYINPLDGTYPPGTEQNPANPNPSLEESQPLNTFMFQGNRFRNRQGALTVYTGTFTQGTDPVKTYYQYTPVSSRAMYDAYWNGKFRDCAFHSGFNEDPFVCEYQGQSSDLPQPPANAGGESGGGSGGG SEGGGSEGGGSEGGGSEGGGSGGGSGSGSEQ ID NO: 20 = MAETVESCLAKPHTENSFTNVWKEGDSRYANYEGCLWNAGGVVVCTGDETQCYGHWVPIGLAIPENEGGGSEGGGSEGGGSEGGGTKPPEYGDTPIPGYTYINPLDGTYPPGTEQNPANPNPSLEESQPLNTFMFQNNRFRNVNGVLIVYTGTFTQGTDPVKTYYQYTPVSSRAMYDAYWNGKFRDCAFHSGFNEDPFVCEYQGQSSDLPQPPANAGGESGGGSGGG SEGGGSEGGGSEGGGSEGGGSGGGSGSGSEQ ID NO: 21 = MAETVESCLAKPHTENSFTNVWKEGDSRYANYEGCLWNAGGVVVCTGDETQCYGHWVPIGLAIPENEGGGSEGGGSEGGGSEGGGTKPPEYGDTPIPGYTYINPLDGTYPPGTEQNPANPNPSLEESQPLNTFMFQGNRFRARQGALTVYTGTFTQGTDPVKTYYQYTPVSSRAMYDAYWNGKFRDCAFHSGFNEDPFVCEYQGQSSDLPQPPANAGGESGGGSGGG SEGGGSEGGGSEGGGSEGGGSGGGSGSGSEQ ID NO: 22 = MAETVESCLAKPHTENSFTNVWKEGDSRYANYEGCLWNAGGVVVCTGDETQCYGHWVPIGLAIPENEGGGSEGGGSEGGGSEGGGTKPPEYGDTPIPGYTYINPLDGTYPPGTEQNPANPNPSLEESQPLNTFMFQNNRFRAVNGVLTVYTGTFTQGTDPVKTYYQYTPVSSRAMYDAYWNGKFRDCAFHSGFNEDPFVCEYQGQSSDLPQPPANAGGESGGGSGGG SEGGGSEGGGSEGGGSEGGGSGGGSGSGSEQ ID NO: 23 = MAETVESCLAKPHTENSFTNVWKEGDSRYANYEGCLWNAGGVVVCTGDEHQCYGTWVPIGLAIPENEGGGSEGGGSEGGGSEGGGTKPPEYGDTPIPGYTYINPLDGTYPPGTEQNPANPNPSLEESQPLNTFMFQGNRFRARQGALTVYTGTFTQGTDPVKTYYQYTPVSSRAMYDAYWNGKFRDCAFHSGFNEDPFVCEYQGQSSDLPQPPANAGGESGGGSGGG SEGGGSEGGGSEGGGSEGGGSGGGSGSGSEQ ID NO: 24 = MAETVESCLAKPHTENSFTNVWKEGDSRYANYEGCLWNAGGVVVCTGDEHQCYGTWVPIGLAIPENEGGGSEGGGSEGGGSEGGGTKPPEYGDTPIPGYTYINPLDGTYPPGTEQNPANPNPSLEESQPLNTFMFQNNRFRAVNGVLTVYTGTFTQGTDPVKTYYQYTPVSSRAMYDAYWNGKFRDCAFHSGFNEDPFVCEYQGQSSDLPQPPANAGGESGGGSGGG SEGGGSEGGGSEGGGSEGGGSGGGSGSGSEQ ID NO: 25 = MAETVESCLAKPHTENSFTNVWKEGDSRYANYEGCLWNAGGVVVCTGDEHQCYGTWVPIGLAIPENEGGGSEGGGSEGGGSEGGGTKPPEYGDTPIPGYTYINPLDGTYPPGTEQNPANPNPSLEESQPLNTFMFQGNRFRNRQGALTVYTGTFTQGTDPVKTYYQYTPVSSRAMYDAYWNGKFRDCAFHSGFNEDPFVCEYQGQSSDLPQPPANAGGESGGGSGGG SEGGGSEGGGSEGGGSEGGGSGGGSGSGSEQ ID NO: 26 = MAETVESCLAKPHTENSFTNVWKEGDSRYANYEGCLWAAGGVVVCTGDEHQCYGTWVPIGLAIPENEGGGSEGGGSEGGGSEGGGTKPPEYGDTPIPGYTYINPLDGTYPPGTEQNPANPNPSLEESQPLNTFMFQGNRFRNRQGALTVYTGTFTQGTDPVKTYYQYTPVSSRAMYDAYWNGKFRDCAFHSGFNEDPFVCEYQGQSSDLPQPPANAGGESGGGSGGG SEGGGSEGGGSEGGGSEGGGSGGGSGSGSEQ ID NO: 27 = GGGGS; SEQ ID NO: 28 = GGGS SEQ ID NO: 29 =MAETVESCLAKPHTENSFTNVWKEGDSRYANYEGCLWNAGGVVVCTGDETQCYGHWVPIGLAIPENEGGGSEGGGSEGGGSEGGGTKPPEYGDTPIPGYTYINPLDGTYPPGTEQNPANPNPSLEESQPLNTFMFQGNRFRNRQGALTVYTGTFTQGTDPVKTYYQYTPVSSRAMYDAYWNGKFRDCAFHSGFNEDPFVCEYQGQSSDLPQPPANAGGESGGGSGGGSEGGGSEGGGSEGGGSEGGGSGGGSGSGARSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNVVYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR WQQGNVFSCSVMHEALHNHYTQKSLSLSPGKSEQ ID NO: 30 = MAETVESCLAKPHTENSFTNVWKEGDSRYANYEGCLWNAGGVVVCTGDETQCYGHWVPIGLAIPENEGGGSEGGGSEGGGSEGGGTKPPEYGDTPIPGYTYINPLDGTYPPGTEQNPANPNPSLEESQPLNTFMFQNNRFRNVNGVLTVYTGTFTQGTDPVKTYYQYTPVSSRAMYDAYWNGKFRDCAFHSGFNEDPFVCEYQGQSSDLPQPPANAGGESGGGSGGGSEGGGSEGGGSEGGGSEGGGSGGGSGSGARSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNVVYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR WQQGNVFSCSVMHEALHNHYTQKSLSLSPGKSEQ ID NO: 31 = MAETVESCLAKPHTENSFTNVWKEGDSRYANYEGCLWNAGGVVVCTGDETQCYGHWVPIGLAIPENEGGGSEGGGSEGGGSEGGGTKPPEYGDTPIPGYTYINPLDGTYPPGTEQNPANPNPSLEESQPLNTFMFQGNRFRARQGALTVYTGTFTQGTDPVKTYYQYTPVSSRAMYDAYWNGKFRDCAFHSGFNEDPFVCEYQGQSSDLPQPPANAGGESGGGSGGGSEGGGSEGGGSEGGGSEGGGSGGGSGSGARSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNVVYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR WQQGNVFSCSVMHEALHNHYTQKSLSLSPGKSEQ ID NO: 32 = MAETVESCLAKPHTENSFTNVWKEGDSRYANYEGCLWNAGGVVVCTGDETQCYGHWVPIGLAIPENEGGGSEGGGSEGGGSEGGGTKPPEYGDTPIPGYTYINPLDGTYPPGTEQNPANPNPSLEESQPLNTFMFQNNRFRAVNGVLTVYTGTFTQGTDPVKTYYQYTPVSSRAMYDAYWNGKFRDCAFHSGFNEDPFVCEYQGQSSDLPQPPANAGGESGGGSGGGSEGGGSEGGGSEGGGSEGGGSGGGSGSGARSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNVVYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR WQQGNVFSCSVMHEALHNHYTQKSLSLSPGKSEQ ID NO: 33 = MAETVESCLAKPHTENSFTNVWKEGDSRYANYEGCLWNAGGVVVCTGDEHQCYGTWVPIGLAIPENEGGGSEGGGSEGGGSEGGGTKPPEYGDTPIPGYTYINPLDGTYPPGTEQNPANPNPSLEESQPLNTFMFQGNRFRARQGALTVYTGTFTQGTDPVKTYYQYTPVSSRAMYDAYWNGKFRDCAFHSGFNEDPFVCEYQGQSSDLPQPPANAGGESGGGSGGGSEGGGSEGGGSEGGGSEGGGSGGGSGSGARSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNVVYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR WQQGNVFSCSVMHEALHNHYTQKSLSLSPGKSEQ ID NO: 34 = MAETVESCLAKPHTENSFTNVWKEGDSRYANYEGCLWNAGGVVVCTGDEHQCYGTWVPIGLAIPENEGGGSEGGGSEGGGSEGGGTKPPEYGDTPIPGYTYINPLDGTYPPGTEQNPANPNPSLEESQPLNTFMFQNNRFRAVNGVLTVYTGTFTQGTDPVKTYYQYTPVSSRAMYDAYWNGKFRDCAFHSGFNEDPFVCEYQGQSSDLPQPPANAGGESGGGSGGGSEGGGSEGGGSEGGGSEGGGSGGGSGSGARSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNVVYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR WQQGNVFSCSVMHEALHNHYTQKSLSLSPGKSEQ ID NO: 35 = MAETVESCLAKPHTENSFTNVWKEGDSRYANYEGCLWNAGGVVVCTGDEHQCYGTWVPIGLAIPENEGGGSEGGGSEGGGSEGGGTKPPEYGDTPIPGYTYINPLDGTYPPGTEQNPANPNPSLEESQPLNTFMFQGNRFRNRQGALTVYTGTFTQGTDPVKTYYQYTPVSSRAMYDAYWNGKFRDCAFHSGFNEDPFVCEYQGQSSDLPQPPANAGGESGGGSGGGSEGGGSEGGGSEGGGSEGGGSGGGSGSGARSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNVVYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR WQQGNVFSCSVMHEALHNHYTQKSLSLSPGKSEQ ID NO: 36 = MAETVESCLAKPHTENSFTNVWKEGDSRYANYEGCLWAAGGVVVCTGDEHQCYGTWVPIGLAIPENEGGGSEGGGSEGGGSEGGGTKPPEYGDTPIPGYTYINPLDGTYPPGTEQNPANPNPSLEESQPLNTFMFQGNRFRNRQGALTVYTGTFTQGTDPVKTYYQYTPVSSRAMYDAYWNGKFRDCAFHSGFNEDPFVCEYQGQSSDLPQPPANAGGESGGGSGGGSEGGGSEGGGSEGGGSEGGGSGGGSGSGARSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNVVYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR WQQGNVFSCSVMHEALHNHYTQKSLSLSPGKSEQ ID NO: 37 = ATGGCCGAAACCGTGGAATCATGTCTGGCGAAGCCCCATACCGAGAACTCCTTCACCAACGTCTGGAAAGAGGGCGACAGCCGCTACGCCAACTACGAGGGCTGCCTGTGGAACGCCGGTGGAGTGGTCGTCTGCACCGGGGATGAGACTCAGTGCTACGGACACTGGGTGCCTATCGGACTGGCCATTCCCGAGAACGAGGGGGGTGGTAGCGAAGGCGGCGGATCGGAAGGCGGAGGATCTGAGGGAGGGGGAACCAAGCCTCCGGAATACGGCGACACTCCGATCCCCGGGTATACGTACATCAATCCGCTGGACGGGACCTACCCGCCTGGAACTGAGCAGAACCCGGCCAACCCAAACCCTAGCCTCGAGGAATCCCAGCCGTTGAACACCTTCATGTTCCAAGGGAACCGCTTCAGGAACAGACAGGGAGCGCTGACCGTGTACACTGGCACCTTCACACAAGGCACCGACCCCGTCAAGACCTACTACCAGTACACTCCTGTGTCCTCGCGGGCTATGTACGATGCGTACTGGAATGGGAAGTTTCGGGACTGCGCTTTCCACTCCGGCTTCAACGAGGATCCATTCGTGTGCGAATATCAGGGCCAGAGCTCCGACCTCCCCCAACCCCCTGCAAACGCCGGCGGAGAATCCGGAGGGGGATCAGGAGGCGGAAGCGAAGGGGGTGGATCCGAAGGAGGCGGATCCGAGGGTGGAGGCTCCGAAGGGGGAGGCTCTGGTGGTGGCTCCGGAT CGGGA SEQ ID NO: 38 =ATGGCCGAAACCGTGGAATCATGTCTGGCGAAGCCCCATACCGAGAACTCCTTCACCAACGTCTGGAAAGAGGGCGACAGCCGCTACGCCAACTACGAGGGCTGCCTGTGGAACGCCGGTGGAGTGGTCGTCTGCACCGGGGATGAGACTCAGTGCTACGGACACTGGGTGCCTATCGGACTGGCCATTCCCGAGAACGAGGGGGGTGGTAGCGAAGGCGGCGGATCGGAAGGCGGAGGATCTGAGGGAGGGGGAACCAAGCCTCCGGAATACGGCGACACTCCGATCCCCGGGTATACGTACATCAATCCGCTGGACGGGACCTACCCGCCTGGAACTGAGCAGAACCCGGCCAACCCAAACCCTAGCCTCGAGGAATCCCAGCCGTTGAACACCTTCATGTTCCAAAACAACCGCTTCAGGAACGTGAACGGAGTGCTGACCGTGTACACTGGCACCTTCACACAAGGCACCGACCCCGTCAAGACCTACTACCAGTACACTCCTGTGTCCTCGCGGGCTATGTACGATGCGTACTGGAATGGGAAGTTTCGGGACTGCGCTTTCCACTCCGGCTTCAACGAGGATCCATTCGTGTGCGAATATCAGGGCCAGAGCTCCGACCTCCCCCAACCCCCTGCAAACGCCGGCGGAGAATCCGGAGGGGGATCAGGAGGCGGAAGCGAAGGGGGTGGATCCGAAGGAGGCGGATCCGAGGGTGGAGGCTCCGAAGGGGGAGGCTCTGGTGGTGGCTCCGGAT CGGGA SEQ ID NO: 39 =ATGGCCGAAACCGTGGAATCATGTCTGGCGAAGCCCCATACCGAGAACTCCTTCACCAACGTCTGGAAAGAGGGCGACAGCCGCTACGCCAACTACGAGGGCTGCCTGTGGAACGCCGGTGGAGTGGTCGTCTGCACCGGGGATGAGACTCAGTGCTACGGACACTGGGTGCCTATCGGACTGGCCATTCCCGAGAACGAGGGGGGTGGTAGCGAAGGCGGCGGATCGGAAGGCGGAGGATCTGAGGGAGGGGGAACCAAGCCTCCGGAATACGGCGACACTCCGATCCCCGGGTATACGTACATCAATCCGCTGGACGGGACCTACCCGCCTGGAACTGAGCAGAACCCGGCCAACCCAAACCCTAGCCTCGAGGAATCCCAGCCGTTGAACACCTTCATGTTCCAAGGGAACCGCTTCAGGGCTAGACAGGGAGCGCTGACCGTGTACACTGGCACCTTCACACAAGGCACCGACCCCGTCAAGACCTACTACCAGTACACTCCTGTGTCCTCGCGGGCTATGTACGATGCGTACTGGAATGGGAAGTTTCGGGACTGCGCTTTCCACTCCGGCTTCAACGAGGATCCATTCGTGTGCGAATATCAGGGCCAGAGCTCCGACCTCCCCCAACCCCCTGCAAACGCCGGCGGAGAATCCGGAGGGGGATCAGGAGGCGGAAGCGAAGGGGGTGGATCCGAAGGAGGCGGATCCGAGGGTGGAGGCTCCGAAGGGGGAGGCTCTGGTGGTGGCTCCGGAT CGGGA SEQ ID NO: 40 =ATGGCCGAAACCGTGGAATCATGTCTGGCGAAGCCCCATACCGAGAACTCCTTCACCAACGTCTGGAAAGAGGGCGACAGCCGCTACGCCAACTACGAGGGCTGCCTGTGGAACGCCGGTGGAGTGGTCGTCTGCACCGGGGATGAGACTCAGTGCTACGGACACTGGGTGCCTATCGGACTGGCCATTCCCGAGAACGAGGGGGGTGGTAGCGAAGGCGGCGGATCGGAAGGCGGAGGATCTGAGGGAGGGGGAACCAAGCCTCCGGAATACGGCGACACTCCGATCCCCGGGTATACGTACATCAATCCGCTGGACGGGACCTACCCGCCTGGAACTGAGCAGAACCCGGCCAACCCAAACCCTAGCCTCGAGGAATCCCAGCCGTTGAACACCTTCATGTTCCAAAACAACCGCTTCAGGGCCGTGAACGGAGTGCTGACCGTGTACACTGGCACCTTCACACAAGGCACCGACCCCGTCAAGACCTACTACCAGTACACTCCTGTGTCCTCGCGGGCTATGTACGATGCGTACTGGAATGGGAAGTTTCGGGACTGCGCTTTCCACTCCGGCTTCAACGAGGATCCATTCGTGTGCGAATATCAGGGCCAGAGCTCCGACCTCCCCCAACCCCCTGCAAACGCCGGCGGAGAATCCGGAGGGGGATCAGGAGGCGGAAGCGAAGGGGGTGGATCCGAAGGAGGCGGATCCGAGGGTGGAGGCTCCGAAGGGGGAGGCTCTGGTGGTGGCTCCGGAT CGGGA SEQ ID NO: 41 =ATGGCCGAAACCGTGGAATCATGTCTGGCGAAGCCCCATACCGAGAACTCCTTCACCAACGTCTGGAAAGAGGGCGACAGCCGCTACGCCAACTACGAGGGCTGCCTGTGGAACGCCGGTGGAGTGGTCGTCTGCACCGGGGATGAGCACCAGTGCTACGGAACTTGGGTGCCTATCGGACTGGCCATTCCCGAGAACGAGGGGGGTGGTAGCGAAGGCGGCGGATCGGAAGGCGGAGGATCTGAGGGAGGGGGAACCAAGCCTCCGGAATACGGCGACACTCCGATCCCCGGGTATACGTACATCAATCCGCTGGACGGGACCTACCCGCCTGGAACTGAGCAGAACCCGGCCAACCCAAACCCTAGCCTCGAGGAATCCCAGCCGTTGAACACCTTCATGTTCCAAGGGAACCGCTTCAGGGCTAGACAGGGAGCGCTGACCGTGTACACTGGCACCTTCACACAAGGCACCGACCCCGTCAAGACCTACTACCAGTACACTCCTGTGTCCTCGCGGGCTATGTACGATGCGTACTGGAATGGGAAGTTTCGGGACTGCGCTTTCCACTCCGGCTTCAACGAGGATCCATTCGTGTGCGAATATCAGGGCCAGAGCTCCGACCTCCCCCAACCCCCTGCAAACGCCGGCGGAGAATCCGGAGGGGGATCAGGAGGCGGAAGCGAAGGGGGTGGATCCGAAGGAGGCGGATCCGAGGGTGGAGGCTCCGAAGGGGGAGGCTCTGGTGGTGGCTCCGGAT CGGGA SEQ ID NO: 42 =ATGGCCGAAACCGTGGAATCATGTCTGGCGAAGCCCCATACCGAGAACTCCTTCACCAACGTCTGGAAAGAGGGCGACAGCCGCTACGCCAACTACGAGGGCTGCCTGTGGAACGCCGGTGGAGTGGTCGTCTGCACCGGGGATGAGCACCAGTGCTACGGAACTTGGGTGCCTATCGGACTGGCCATTCCCGAGAACGAGGGGGGTGGTAGCGAAGGCGGCGGATCGGAAGGCGGAGGATCTGAGGGAGGGGGAACCAAGCCTCCGGAATACGGCGACACTCCGATCCCCGGGTATACGTACATCAATCCGCTGGACGGGACCTACCCGCCTGGAACTGAGCAGAACCCGGCCAACCCAAACCCTAGCCTCGAGGAATCCCAGCCGTTGAACACCTTCATGTTCCAAAACAACCGCTTCAGGGCCGTGAACGGAGTGCTGACCGTGTACACTGGCACCTTCACACAAGGCACCGACCCCGTCAAGACCTACTACCAGTACACTCCTGTGTCCTCGCGGGCTATGTACGATGCGTACTGGAATGGGAAGTTTCGGGACTGCGCTTTCCACTCCGGCTTCAACGAGGATCCATTCGTGTGCGAATATCAGGGCCAGAGCTCCGACCTCCCCCAACCCCCTGCAAACGCCGGCGGAGAATCCGGAGGGGGATCAGGAGGCGGAAGCGAAGGGGGTGGATCCGAAGGAGGCGGATCCGAGGGTGGAGGCTCCGAAGGGGGAGGCTCTGGTGGTGGCTCCGGAT CGGGA SEQ ID NO: 43 =ATGGCCGAAACCGTGGAATCATGTCTGGCGAAGCCCCATACCGAGAACTCCTTCACCAACGTCTGGAAAGAGGGCGACAGCCGCTACGCCAACTACGAGGGCTGCCTGTGGAACGCCGGTGGAGTGGTCGTCTGCACCGGGGATGAGCATCAGTGCTACGGAACCTGGGTGCCTATCGGACTGGCCATTCCCGAGAACGAGGGGGGTGGTAGCGAAGGCGGCGGATCGGAAGGCGGAGGATCTGAGGGAGGGGGAACCAAGCCTCCGGAATACGGCGACACTCCGATCCCCGGGTATACGTACATCAATCCGCTGGACGGGACCTACCCGCCTGGAACTGAGCAGAACCCGGCCAACCCAAACCCTAGCCTCGAGGAATCCCAGCCGTTGAACACCTTCATGTTCCAAGGGAACCGCTTCAGGAACAGACAGGGAGCGCTGACCGTGTACACTGGCACCTTCACACAAGGCACCGACCCCGTCAAGACCTACTACCAGTACACTCCTGTGTCCTCGCGGGCTATGTACGATGCGTACTGGAATGGGAAGTTTCGGGACTGCGCTTTCCACTCCGGCTTCAACGAGGATCCATTCGTGTGCGAATATCAGGGCCAGAGCTCCGACCTCCCCCAACCCCCTGCAAACGCCGGCGGAGAATCCGGAGGGGGATCAGGAGGCGGAAGCGAAGGGGGTGGATCCGAAGGAGGCGGATCCGAGGGTGGAGGCTCCGAAGGGGGAGGCTCTGGTGGTGGCTCCGGAT CGGGA SEQ ID NO: 44 =ATGGCCGAAACCGTGGAATCATGTCTGGCGAAGCCCCATACCGAGAACTCCTTCACCAACGTCTGGAAAGAGGGCGACAGCCGCTACGCCAACTACGAGGGCTGCCTGTGGGCCGCCGGTGGAGTGGTCGTCTGCACCGGGGATGAGCATCAGTGCTACGGAACCTGGGTGCCTATCGGACTGGCCATTCCCGAGAACGAGGGGGGTGGTAGCGAAGGCGGCGGATCGGAAGGCGGAGGATCTGAGGGAGGGGGAACCAAGCCTCCGGAATACGGCGACACTCCGATCCCCGGGTATACGTACATCAATCCGCTGGACGGGACCTACCCGCCTGGAACTGAGCAGAACCCGGCCAACCCAAACCCTAGCCTCGAGGAATCCCAGCCGTTGAACACCTTCATGTTCCAAGGGAACCGCTTCAGGAACAGACAGGGAGCGCTGACCGTGTACACTGGCACCTTCACACAAGGCACCGACCCCGTCAAGACCTACTACCAGTACACTCCTGTGTCCTCGCGGGCTATGTACGATGCGTACTGGAATGGGAAGTTTCGGGACTGCGCTTTCCACTCCGGCTTCAACGAGGATCCATTCGTGTGCGAATATCAGGGCCAGAGCTCCGACCTCCCCCAACCCCCTGCAAACGCCGGCGGAGAATCCGGAGGGGGATCAGGAGGCGGAAGCGAAGGGGGTGGATCCGAAGGAGGCGGATCCGAGGGTGGAGGCTCCGAAGGGGGAGGCTCTGGTGGTGGCTCCGGAT CGGGA SEQ ID NO: 45 =ATGGCCGAAACCGTGGAATCATGTCTGGCGAAGCCCCATACCGAGAACTCCTTCACCAACGTCTGGAAAGAGGGCGACAGCCGCTACGCCAACTACGAGGGCTGCCTGTGGAACGCCGGTGGAGTGGTCGTCTGCACCGGGGATGAGACTCAGTGCTACGGACACTGGGTGCCTATCGGACTGGCCATTCCCGAGAACGAGGGGGGTGGTAGCGAAGGCGGCGGATCGGAAGGCGGAGGATCTGAGGGAGGGGGAACCAAGCCTCCGGAATACGGCGACACTCCGATCCCCGGGTATACGTACATCAATCCGCTGGACGGGACCTACCCGCCTGGAACTGAGCAGAACCCGGCCAACCCAAACCCTAGCCTCGAGGAATCCCAGCCGTTGAACACCTTCATGTTCCAAGGGAACCGCTTCAGGAACAGACAGGGAGCGCTGACCGTGTACACTGGCACCTTCACACAAGGCACCGACCCCGTCAAGACCTACTACCAGTACACTCCTGTGTCCTCGCGGGCTATGTACGATGCGTACTGGAATGGGAAGTTTCGGGACTGCGCTTTCCACTCCGGCTTCAACGAGGATCCATTCGTGTGCGAATATCAGGGCCAGAGCTCCGACCTCCCCCAACCCCCTGCAAACGCCGGCGGAGAATCCGGAGGGGGATCAGGAGGCGGAAGCGAAGGGGGTGGATCCGAAGGAGGCGGATCCGAGGGTGGAGGCTCCGAAGGGGGAGGCTCTGGTGGTGGCTCCGGATCGGGAGCCAGATCTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCACGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGT CTCCGGGTAAATGA SEQ ID NO: 46 =ATGGCCGAAACCGTGGAATCATGTCTGGCGAAGCCCCATACCGAGAACTCCTTCACCAACGTCTGGAAAGAGGGCGACAGCCGCTACGCCAACTACGAGGGCTGCCTGTGGAACGCCGGTGGAGTGGTCGTCTGCACCGGGGATGAGACTCAGTGCTACGGACACTGGGTGCCTATCGGACTGGCCATTCCCGAGAACGAGGGGGGTGGTAGCGAAGGCGGCGGATCGGAAGGCGGAGGATCTGAGGGAGGGGGAACCAAGCCTCCGGAATACGGCGACACTCCGATCCCCGGGTATACGTACATCAATCCGCTGGACGGGACCTACCCGCCTGGAACTGAGCAGAACCCGGCCAACCCAAACCCTAGCCTCGAGGAATCCCAGCCGTTGAACACCTTCATGTTCCAAAACAACCGCTTCAGGAACGTGAACGGAGTGCTGACCGTGTACACTGGCACCTTCACACAAGGCACCGACCCCGTCAAGACCTACTACCAGTACACTCCTGTGTCCTCGCGGGCTATGTACGATGCGTACTGGAATGGGAAGTTTCGGGACTGCGCTTTCCACTCCGGCTTCAACGAGGATCCATTCGTGTGCGAATATCAGGGCCAGAGCTCCGACCTCCCCCAACCCCCTGCAAACGCCGGCGGAGAATCCGGAGGGGGATCAGGAGGCGGAAGCGAAGGGGGTGGATCCGAAGGAGGCGGATCCGAGGGTGGAGGCTCCGAAGGGGGAGGCTCTGGTGGTGGCTCCGGATCGGGAGCCAGATCTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCACGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGT CTCCGGGTAAATGA SEQ ID NO: 47 =ATGGCCGAAACCGTGGAATCATGTCTGGCGAAGCCCCATACCGAGAACTCCTTCACCAACGTCTGGAAAGAGGGCGACAGCCGCTACGCCAACTACGAGGGCTGCCTGTGGAACGCCGGTGGAGTGGTCGTCTGCACCGGGGATGAGACTCAGTGCTACGGACACTGGGTGCCTATCGGACTGGCCATTCCCGAGAACGAGGGGGGTGGTAGCGAAGGCGGCGGATCGGAAGGCGGAGGATCTGAGGGAGGGGGAACCAAGCCTCCGGAATACGGCGACACTCCGATCCCCGGGTATACGTACATCAATCCGCTGGACGGGACCTACCCGCCTGGAACTGAGCAGAACCCGGCCAACCCAAACCCTAGCCTCGAGGAATCCCAGCCGTTGAACACCTTCATGTTCCAAGGGAACCGCTTCAGGGCTAGACAGGGAGCGCTGACCGTGTACACTGGCACCTTCACACAAGGCACCGACCCCGTCAAGACCTACTACCAGTACACTCCTGTGTCCTCGCGGGCTATGTACGATGCGTACTGGAATGGGAAGTTTCGGGACTGCGCTTTCCACTCCGGCTTCAACGAGGATCCATTCGTGTGCGAATATCAGGGCCAGAGCTCCGACCTCCCCCAACCCCCTGCAAACGCCGGCGGAGAATCCGGAGGGGGATCAGGAGGCGGAAGCGAAGGGGGTGGATCCGAAGGAGGCGGATCCGAGGGTGGAGGCTCCGAAGGGGGAGGCTCTGGTGGTGGCTCCGGATCGGGAGCCAGATCTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCACGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGT CTCCGGGTAAATGA SEQ ID NO: 48 =ATGGCCGAAACCGTGGAATCATGTCTGGCGAAGCCCCATACCGAGAACTCCTTCACCAACGTCTGGAAAGAGGGCGACAGCCGCTACGCCAACTACGAGGGCTGCCTGTGGAACGCCGGTGGAGTGGTCGTCTGCACCGGGGATGAGACTCAGTGCTACGGACACTGGGTGCCTATCGGACTGGCCATTCCCGAGAACGAGGGGGGTGGTAGCGAAGGCGGCGGATCGGAAGGCGGAGGATCTGAGGGAGGGGGAACCAAGCCTCCGGAATACGGCGACACTCCGATCCCCGGGTATACGTACATCAATCCGCTGGACGGGACCTACCCGCCTGGAACTGAGCAGAACCCGGCCAACCCAAACCCTAGCCTCGAGGAATCCCAGCCGTTGAACACCTTCATGTTCCAAAACAACCGCTTCAGGGCCGTGAACGGAGTGCTGACCGTGTACACTGGCACCTTCACACAAGGCACCGACCCCGTCAAGACCTACTACCAGTACACTCCTGTGTCCTCGCGGGCTATGTACGATGCGTACTGGAATGGGAAGTTTCGGGACTGCGCTTTCCACTCCGGCTTCAACGAGGATCCATTCGTGTGCGAATATCAGGGCCAGAGCTCCGACCTCCCCCAACCCCCTGCAAACGCCGGCGGAGAATCCGGAGGGGGATCAGGAGGCGGAAGCGAAGGGGGTGGATCCGAAGGAGGCGGATCCGAGGGTGGAGGCTCCGAAGGGGGAGGCTCTGGTGGTGGCTCCGGATCGGGAGCCAGATCTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCACGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGT CTCCGGGTAAATGA SEQ ID NO: 49 =ATGGCCGAAACCGTGGAATCATGTCTGGCGAAGCCCCATACCGAGAACTCCTTCACCAACGTCTGGAAAGAGGGCGACAGCCGCTACGCCAACTACGAGGGCTGCCTGTGGAACGCCGGTGGAGTGGTCGTCTGCACCGGGGATGAGCACCAGTGCTACGGAACTTGGGTGCCTATCGGACTGGCCATTCCCGAGAACGAGGGGGGTGGTAGCGAAGGCGGCGGATCGGAAGGCGGAGGATCTGAGGGAGGGGGAACCAAGCCTCCGGAATACGGCGACACTCCGATCCCCGGGTATACGTACATCAATCCGCTGGACGGGACCTACCCGCCTGGAACTGAGCAGAACCCGGCCAACCCAAACCCTAGCCTCGAGGAATCCCAGCCGTTGAACACCTTCATGTTCCAAGGGAACCGCTTCAGGGCTAGACAGGGAGCGCTGACCGTGTACACTGGCACCTTCACACAAGGCACCGACCCCGTCAAGACCTACTACCAGTACACTCCTGTGTCCTCGCGGGCTATGTACGATGCGTACTGGAATGGGAAGTTTCGGGACTGCGCTTTCCACTCCGGCTTCAACGAGGATCCATTCGTGTGCGAATATCAGGGCCAGAGCTCCGACCTCCCCCAACCCCCTGCAAACGCCGGCGGAGAATCCGGAGGGGGATCAGGAGGCGGAAGCGAAGGGGGTGGATCCGAAGGAGGCGGATCCGAGGGTGGAGGCTCCGAAGGGGGAGGCTCTGGTGGTGGCTCCGGATCGGGAGCCAGATCTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCACGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGT CTCCGGGTAAATGA\ SEQ ID NO: 50 =ATGGCCGAAACCGTGGAATCATGTCTGGCGAAGCCCCATACCGAGAACTCCTTCACCAACGTCTGGAAAGAGGGCGACAGCCGCTACGCCAACTACGAGGGCTGCCTGTGGAACGCCGGTGGAGTGGTCGTCTGCACCGGGGATGAGCACCAGTGCTACGGAACTTGGGTGCCTATCGGACTGGCCATTCCCGAGAACGAGGGGGGTGGTAGCGAAGGCGGCGGATCGGAAGGCGGAGGATCTGAGGGAGGGGGAACCAAGCCTCCGGAATACGGCGACACTCCGATCCCCGGGTATACGTACATCAATCCGCTGGACGGGACCTACCCGCCTGGAACTGAGCAGAACCCGGCCAACCCAAACCCTAGCCTCGAGGAATCCCAGCCGTTGAACACCTTCATGTTCCAAAACAACCGCTTCAGGGCCGTGAACGGAGTGCTGACCGTGTACACTGGCACCTTCACACAAGGCACCGACCCCGTCAAGACCTACTACCAGTACACTCCTGTGTCCTCGCGGGCTATGTACGATGCGTACTGGAATGGGAAGTTTCGGGACTGCGCTTTCCACTCCGGCTTCAACGAGGATCCATTCGTGTGCGAATATCAGGGCCAGAGCTCCGACCTCCCCCAACCCCCTGCAAACGCCGGCGGAGAATCCGGAGGGGGATCAGGAGGCGGAAGCGAAGGGGGTGGATCCGAAGGAGGCGGATCCGAGGGTGGAGGCTCCGAAGGGGGAGGCTCTGGTGGTGGCTCCGGATCGGGAGCCAGATCTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCACGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGT CTCCGGGTAAATGA SEQ ID NO: 51 =ATGGCCGAAACCGTGGAATCATGTCTGGCGAAGCCCCATACCGAGAACTCCTTCACCAACGTCTGGAAAGAGGGCGACAGCCGCTACGCCAACTACGAGGGCTGCCTGTGGAACGCCGGTGGAGTGGTCGTCTGCACTGGGGATGAGCACCAGTGCTACGGAACCTGGGTGCCTATCGGACTGGCCATTCCCGAGAACGAGGGGGGTGGTAGCGAAGGCGGCGGATCGGAAGGCGGAGGATCTGAGGGAGGGGGAACCAAGCCTCCGGAATACGGCGACACTCCGATCCCCGGGTATACGTACATCAATCCGCTGGACGGGACCTACCCGCCTGGAACTGAGCAGAACCCGGCCAACCCAAACCCTAGCCTCGAGGAATCCCAGCCGTTGAACACCTTCATGTTCCAAGGGAACCGCTTCAGGAACAGACAGGGAGCGCTGACCGTGTACACTGGCACCTTCACACAAGGCACCGACCCCGTCAAGACCTACTACCAGTACACTCCTGTGTCCTCGCGGGCTATGTACGATGCGTACTGGAATGGGAAGTTTCGGGACTGCGCTTTCCACTCCGGCTTCAACGAGGATCCATTCGTGTGCGAATATCAGGGCCAGAGCTCCGACCTCCCCCAACCCCCTGCAAACGCCGGCGGAGAATCCGGAGGGGGATCAGGAGGCGGAAGCGAAGGGGGTGGATCCGAAGGAGGCGGATCCGAGGGTGGAGGCTCCGAAGGGGGAGGCTCTGGTGGTGGCTCCGGATCGGGAGCCAGATCTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCACGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGT CTCCGGGTAAATGA SEQ ID NO: 52 =ATGGCCGAAACCGTGGAATCATGTCTGGCGAAGCCCCATACCGAGAACTCCTTCACCAACGTCTGGAAAGAGGGCGACAGCCGCTACGCCAACTACGAGGGCTGCCTGTGGGCCGCCGGTGGAGTGGTCGTCTGCACTGGGGATGAGCACCAGTGCTACGGAACCTGGGTGCCTATCGGACTGGCCATTCCCGAGAACGAGGGGGGTGGTAGCGAAGGCGGCGGATCGGAAGGCGGAGGATCTGAGGGAGGGGGAACCAAGCCTCCGGAATACGGCGACACTCCGATCCCCGGGTATACGTACATCAATCCGCTGGACGGGACCTACCCGCCTGGAACTGAGCAGAACCCGGCCAACCCAAACCCTAGCCTCGAGGAATCCCAGCCGTTGAACACCTTCATGTTCCAAGGGAACCGCTTCAGGAACAGACAGGGAGCGCTGACCGTGTACACTGGCACCTTCACACAAGGCACCGACCCCGTCAAGACCTACTACCAGTACACTCCTGTGTCCTCGCGGGCTATGTACGATGCGTACTGGAATGGGAAGTTTCGGGACTGCGCTTTCCACTCCGGCTTCAACGAGGATCCATTCGTGTGCGAATATCAGGGCCAGAGCTCCGACCTCCCCCAACCCCCTGCAAACGCCGGCGGAGAATCCGGAGGGGGATCAGGAGGCGGAAGCGAAGGGGGTGGATCCGAAGGAGGCGGATCCGAGGGTGGAGGCTCCGAAGGGGGAGGCTCTGGTGGTGGCTCCGGATCGGGAGCCAGATCTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCACGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGT CTCCGGGTAAATGA SEQ ID NO: 53 =HHHHHH, SEQ ID NO: 54 = EDGS

Definitions

The terms used in this specification generally have their ordinarymeanings in the art, within the context of this invention and in thespecific context where each term is used. Certain terms are discussedbelow, or elsewhere in the specification, to provide additional guidanceto the practitioner in describing the compositions and methods of theinvention and how to make and use them. The articles “a” and “an” referto one or to more than one (i.e., to at least one) of the grammaticalobject of the article. The term “or” means, and is used interchangeablywith, the term “and/or,” unless context clearly indicates otherwise. Inthis application, the use of the singular includes the plural unlessspecifically stated otherwise. Furthermore, the use of the term“comprising,” as well as other forms, such as “comprises” and“comprised,” are not limiting. Any range described herein will beunderstood to include the endpoints and all values between theendpoints.

The term “g3p” when used alone or in terms such as “g3p-derived” or “g3pfusion” refers to any wild type or recombinant filamentous phage g3pprotein, including fragments, variants, and mutants of g3p that retainthe ability to bind to amyloid. These terms should not be construed aslimited to any particular filamentous bacteriophage g3p.

The terms “filamentous bacteriophage,” “bacteriophage,” and “phage” areused interchangeably herein and include both wild type and recombinantfilamentous bacteriophage.

The term “wild type filamentous bacteriophage” as used herein refers tofilamentous bacteriophage found in nature, filamentous bacteriophagethat have been indicated as “wild type” in any nucleotide or amino acidsequence database, filamentous bacteriophage that are commerciallyavailable and characterized as “wild type,” and filamentousbacteriophage that have acquired non-recombinant mutations relative toany of the foregoing through passaging.

As used herein, the term “domain” means a region of a polypeptide orprotein having some distinctive physical feature or distinctive role,including, for example, an independently folded structure composed of asection of a polypeptide chain. A domain may contain the sequence of thedistinctive physical feature of the polypeptide or it may contain afragment of the physical feature that retains its bindingcharacteristics (e.g., it retains the ability to bind to a seconddomain). A domain may be associated with another domain. For example,the g3p N2 domain binds F-pili and the g3p N1 domain binds ToIA.

As used herein, “general amyloid interaction motif” or “GAIM” refers toa two-domain polypeptide (N1 and N2 domains of g3p) that mediatesamyloid binding using a combination of both hydrophobic and polarresidues lining the inner surfaces of the molecule. The N1 and the N2domains of the GAIM monomer have an asymmetric distribution of aromaticamino acids. The GAIM N2 domain contains 11 Tyrosine (Tyr) residues and1 exposed Tryptophan (Trp) residue; the N1 domain contains 3 Trp and 3Tyr residues. The N1 and N2 domains adopt an inverted horseshoeconformation and are held together in a closed conformation (lockedconformation) by an intricate network of hydrogen bonds (Weininger etal., 2009). A cis-trans isomerization of the prolines in theinter-domain linker leads to progressive breakage of the hydrogen-bondsand partial opening of the two domains. The “opening” rearrangement ofthe N1 and N2 domains of GAIM exposes β-strands 4 and 5 of N1(comprising polar residues) and β-strands 9 and 10 of N2 (comprisingaromatic/hydrophobic residues) and allows for binding to amyloid. SeeFIG. 1A.

As used herein, “control scaffold” or “GAIM scaffold” corresponds to theGAIM-Ig fusion protein PB120, having the amino acid sequence of SEQ IDNO:11. The GAIM portion of the GAIM scaffold has the amino acid sequenceof SEQ ID NO:12. PB120 is derived from PB106, having the amino acidsequence of SEQ ID NO:13. The GAIM portion of PB106 has the amino acidsequence of SEQ ID NO:14.

As used herein, “PB106+EDGS” (“EDGS” disclosed as SEQ ID NO:54)represents an open-conformation polypeptide having the amino acidsequence as provided by SEQ ID NO:15. The GAIM portion of “PB106+EDGS”(“EDGS” disclosed as SEQ ID NO:54) has the amino acid sequence of SEQ IDNO:16.

As used herein, “super-stabilized” GAIM or GAIM fusion refers to theGAIM-Ig fusion PB113, having the amino acid sequence of SEQ ID NO:17.The GAIM portion of PB113 has the amino acid sequence of SEQ ID NO:18.

The terms “GAIM-Ig fusion,” “GAIM-Ig fusion protein,” and “GAIM fusion”are herein used interchangeably and refer to a polypeptide comprising ag3p GAIM domains connected directly to or through a small linker to animmunoglobulin constant region. As shown in FIG. 1B, the Fc region ofthe GAIM-Ig fusions of the invention dimerize, resulting in a complexthat comprises two copies of GAIM attached directly or through a smalllinker to an immunoglobulin constant region. A GAIM fusion of theinvention may further comprising a signal sequence. The presentinvention contemplates GAIM fusions with any immunoglobulin constantregion, for example, the immunoglobulin constant region of IgG (e.g.,IgG1, IgG2, IgG3, or IgG4), IgD, IgA, IgE, or IgM. In some aspects, theGAIM-Ig fusion is an open-stabilized GAIM-Ig fusion. In some aspects,the GAIM-Ig fusion is partially or fully deimmunized.

“GAIM dimer,” as used herein, refers to the two GAIM domains of aGAIM-Ig fusion described herein. A GAIM dimer is graphically representedby FIG. 1C.

The terms “open-stabilized fusion,” “open-stabilized GAIM-Ig fusion,”“open-stabilized variant,” and “open-stabilized GAIM-Ig variant” areused interchangeably herein and refer to a GAIM-Ig fusion comprising atleast one open conformation mutation and at least one stabilizingmutation in the GAIM portion of the fusion. The open-conformationmutation of the open-stabilized variants described herein is asubstitution of SEQ ID NO:3 (DDKTLD; amino acids 24-29 relative to SEQID NO:13 and SEQ ID NO:15) with SEQ ID NO:4 (EGDS). A stabilizingmutation may be an N2-stabilizing mutation, for example, selected fromsubstitution of SEQ ID NO:5 (FQNN; amino acids 137-140 relative to SEQID NO:13; amino acids 135-138 relative to SEQ ID NO:15) with SEQ ID NO:8(FQGN), substitution of SEQ ID NO:6 (RQGA; amino acids 145-148 relativeto SEQ ID NO:13; amino acids 143-146 relative to SEQ ID NO:15) with SEQID NO:9 (VNGV), substitution of SEQ ID NO:7 (QGTDPVK; amino acids158-164 relative to SEQ ID NO:13; amino acids 156-162 relative to SEQ IDNO:15) to SEQ ID NO:10 (QGGK), or a combination thereof. OtherN2-stabilizing mutations are described below. Open-stabilized fusions asdescribed herein may further comprise one or more substitutions,insertions, or deletions. For example, an open-stabilized GAIM-Ig fusionmay be further modified to reduce or eliminate immunogenicity, to removea potential glycosylation site, or to further modulate binding activityor specificity to amyloid protein.

As used herein, a GAIM-Ig fusion protein of the invention that “consistsessentially” of a given amino acid sequence may further include a smalllinker connecting the GAIM domain and Fc domain of the fusion, anN-terminal signal sequence or a fragment thereof, a deletion of theN-terminal methionine (ΔM1) or a deletion of both the N-terminalmethionine and alanine (ΔM1 and ΔA2), and/or a deletion of theC-terminal lysine (K) of the Fc domain of the fusion.

As used herein, a “small linker” refers to a peptide linker up to 25amino acids in length, which connects the GAIM domain and the Fc domainof a GAIM-Ig fusion. As described further below, an exemplary “smalllinker” connecting the GAIM and Fc domains of a GAIM-Ig fusion maycomprise a GS-rich sequence or may comprise the amino acid sequence ARS.

As used herein, a “signal sequence” refers to a short peptide ofapproximately 16 to 30 amino acids present at the N-terminus of apolypeptide of the invention. For example, the signal sequence maycomprise the 18-amino acid N-terminal sequence of GenBank Ref SeqNP_510891.1. A signal sequence is used by a eukaryotic cell to secrete apolypeptide of the invention. It is typically cleaved from thepolypeptide prior to secretion and therefore is typically absent in thesecreted polypeptide.

The term “amyloid” or “amyloid fiber” is used herein as a generic termfor a tertiary structure that is formed by misfolding or aggregation ofany of several different proteins and that comprises an orderedarrangement of β-sheets stacked perpendicular to a fiber axis. Sunde etal., J. Mol Biol. (1997) 273:729-39.

Amyloid, as used herein, can be formed from any of the followingproteins: Androgen receptor; apolipoprotein AI; apolipoprotein AII;apolipoprotein AIV; aposerum amyloid A; Aβ; ABri; ADan; Atrophin-1;atrial natriuretic factor; ataxin; calcitonin; γ-crystallin; cystatin C;fibrinogen; gelsolin; huntingtin; insulin; islet amyloid polypeptide;immunoglobulin kappa light chain; immunoglobulin lambda light chain;kerato-epithelin; keratin; lactahedrin; lactoferrin; lysozyme; lungsurfactant protein C; medin; odontogenic ameloblast-associated protein;prion protein; procalcitonin; prolactin; semenogelin I; serum amyloid A;superoxide dismutase I; β2-microglobulin; TATA box binding protein; tau;transthyretin; and α-synuclein, or to a combination of the above.Amyloid, as used herein, can also be formed from truncated orpost-translationally modified forms of any of the above proteins.“Amyloid” or “amyloid fiber” includes different or multipleconformations or morphologies of amyloid.

As used herein, “toxic oligomer” refers to a small assembly or aggregateof monomers that is typically on-pathway for formation of amyloid.

Exemplary amyloid includes amyloid-β aggregates formed in Alzheimer'sdisease, which comprises beta-amyloid peptide “Aβ,” 39-43 amino acidinternal fragments cleaved from the human amyloid precursor protein(hAPP). Aβ includes truncated and post-translationally modified forms.For example, Aβ40 is a short form of Aβ, and the more fibrillogenicisoform Aβ42 is a long form. Further examples of Aβ include, but are notlimited to, N-truncated Aβ11-42, Aβ11-42-Pyro, Aβ3-42-Pyro, andAβ1-42-E22Q-Dutch mutation. See Levy et al, 1990; Van Broeckhoven et al,1990. Other exemplary amyloid proteins include α-synuclein (associatedwith Parkinson's disease), huntingtin (associated with Huntington'sdisease), tau (associated with Alzheimer's Disease), the abnormalconformation of the prion protein, PrP^(sc), and amyloid associated withvarious amyloidosis diseases, including but not limited to:immunoglobulin light chain (kappa or lambda), transthyretin, gelsolin,and islet amyloid polypeptide. Additional examples are providedthroughout the description and are known to those of skill in the art(see, e.g., Aguzzi (2010), and Eichner and Radford, Mol. Cell (2011)43:8-18). Unless a protein or peptide is specified, use of the terms“amyloid” or “amyloid fibers” should not be construed as limited to anyparticular protein, morphology, disease, or condition.

The term “beta amyloid peptide” is synonymous with “β-amyloid peptide,”βAP,” “βA,” and “Aβ.” All of these terms refer to an amyloid formingpeptide derived from the human amyloid precursor protein (hAPP).

As used herein, “PrP protein,” “PrP,” and “prion,” refer to polypeptidesthat are capable under appropriate conditions of inducing the formationof aggregates responsible for protein misfolding diseases. For example,normal cellular prion protein (PrPc) is converted under appropriateconditions into the corresponding scrapie isoform (PrP^(sc)) that isresponsible for diseases such as, but not limited to, bovine spongiformencephalopathy (BSE) (mad cow disease), feline spongiform encephalopathyof cats, kuru, Creutzfeldt-Jakob Disease (CJD),Gerstmann-Sträussler-Scheinker disease (GSS), and fatal familialinsomnia (FFI).

As used herein, a “disease associated with misfolded and/or aggregatedamyloid protein” includes but is not limited to Alzheimer's disease;early onset Alzheimer's disease; late onset Alzheimer's disease;presymptomatic Alzheimer's disease; AL amyloidosis; amyotrophic lateralsclerosis (ALS); Amyotrophic lateral sclerosis/parkinsonism-dementiacomplex; Argyrophilic grain dementia; Aortic medial amyloidosis; ApoAIamyloidosis; ApoAII amyloidosis; ApoAIV amyloidosis; Atrial amyloidosis;British/Danish dementia; Cataract; Corticobasal degeneration; Cornealamyloidosis associated with trichiasis; cystatin C plaque-relateddisease; cystatin C plaque-related coronary disease; cystatin Cplaque-related kidney disease; cutaneous lichen amyloidosis; Dementiapugilistica; dentatorubral-pallidoluysian atrophy; diffuseneurofibrillary tangles with calcification; dementia with Lewy bodies;Down's syndrome; Familial Amyloidotic Cardiomyopathy (FAC); FamilialAmyloidotic Polyneuropathy (FAP); Familial British dementia; FamiliaDanish dementia; familial encephalopathy; Familial Mediterranean fever;Fibrinogen amyloidosis; Finnish hereditary amyloidosis; Frontotemporaldementia with Parkinsonism; frontotemporal lobar degeneration (FTLDs);frontotemporal lobe dementia; Hallervorden-Spatz disease;Hemodialysis-related amyloidosis; hereditary cerebral amyloidangiopathy; hereditary cerebral hemorrhage with amyloidosis; hereditarylattice corneal dystrophy; Huntington's disease; Icelandic hereditarycerebral amyloid angiopathy; Inclusion-body myositis;Injection-localized amyloidosis; islet amyloid polypeptide amyloidosis;Lysozyme amyloidosis; multiple myeloma; Myotonic dystrophy; Niemann-Pickdisease type C; Non-Guamanian motor neuron disease with neurofibrillarytangles; Parkinson's disease; peripheral amyloidosis; Pick's disease;Pituitary prolactinoma; Postencephalitic parkinsonism; Prion proteincerebral amyloid angiopathy; prion-mediated disease; kuru;Creutzfeldt-Jakob disease (CJD); Gerstmann-Sträussler-Scheinker disease(GSS); fatal familial insomnia (FFI); scrapie; spongiformencephalopathy; pulmonary alveolar proteinosis; Progressive subcorticalgliosis; Progressive supranuclear palsy; Senile Systemic Amyloidosis;serum AA amyloidosis; spinal and bulbar muscular atrophy;spinocerebellar ataxia (SCA1, SCA3, SCA6, or SCA7); Subacute sclerosingpanencephalitis; systemic amyloidosis; familial amyloidosis; wild-typeamyloidosis; Tangle only dementia; and Tauopathies. See, for example,Chiti & Dobson, Annu Rev Biochem (2006) 75:333-66; and Josephs et al.,Acta Neuropathol (2011) 122:137-153. There is a great need to preventand/or reduce amyloid aggregate formation (i.e., misfolded and/oraggregated proteins) to treat or reduce the symptoms or severity ofthese diseases.

As used herein, a polypeptide, composition, formulation, or nucleic acidthat “reduces amyloid” does one or more of the following: inhibitsamyloid formation, causes amyloid disaggregation, causes amyloidremodeling, promotes amyloid clearance, inhibits amyloid aggregation,blocks and/or prevents the formation of toxic oligomers, and/or promotesthe clearance of toxic oligomers.

Polypeptides, nucleic acids or compositions of the invention ordescribed as “disaggregating” or “mediating disaggregation” reduceaggregates that have already formed. Disaggregation can be measured bythe filter trap assay (Wanker et al., Methods Enzymol (1999) 309:375-86)or other methods known in the art. The filter trap assay can be usedboth to detect aggregates and to monitor disaggregation mediated bycompositions of the invention. Disaggregation is detected as decreasedretention of amyloid on the filter, as shown by a decrease in staining,in the presence of increasing concentrations of the disaggregatingagent.

Polypeptides, nucleic acids, or compositions of the invention describedas “protecting neurons from amyloid damage” prevent the accumulation ofnew amyloid and/or prevent the formation of toxic oligomers. Products orcompositions of the invention described as “protecting neurons fromamyloid damage” may be taken prophylactically. Whether or not a productor composition protects neurons from amyloid damage may be measured by aneuronal cell culture cytotoxicity assay as described in WO 2014/055515,hereby incorporated by reference in its entirety.

Polypeptides, nucleic acids, or compositions of the invention describedas “remodeling” amyloid cause partial or complete transformation offibrillar conformers into amorphous aggregates. Remodeling may bemeasured through denaturation studies using urea (e.g., for forms of Aβ)or a sarkosyl solubility assay (e.g., for forms of tau). Increasedremodeling may be detected by the loss or failure of amyloid to bindThioflavin T (ThT), resulting in reduced ThT fluorescence. Remodelingmay also be detected using transmission electron microscopy (TEM).

Polypeptides, nucleic acids, or compositions of the invention describedas “inhibiting amyloid aggregation” partially or completely preventaggregation of amyloid. Inhibition of amyloid aggregation may bemeasured by a ThT fluorescence assay (e.g., lower fluorescenceindicating a lower percentage of amyloid aggregation).

The term “variant” as used herein in conjunction with a bacteriophage,protein, polypeptide, or amino acid sequence (e.g., a GAIM variant),refers to a corresponding substance that contains at least one aminoacid difference (at least one mutation, being a substitution, insertion,or deletion) as compared to the reference substance. In certainembodiments, a “variant” has high amino acid sequence homology and/orconservative amino acid substitutions, deletions, and/or insertions ascompared to the reference sequence. In some embodiments, a variant hasno more than 25, 20, 17, 15, 12, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 aminoacid differences as compared to the reference sequence. A variant asdescribed herein may preserve or increase: amyloid-binding activity,amyloid-binding specificity, and/or protein quality as compared to thereference sequence. A variant as described herein may be deglycosylated.A variant as described herein may reduce or eliminate immunogenicity.

A “conservative substitution” refers to the replacement of a first aminoacid by a second amino acid that does not substantially alter thechemical, physical and/or functional properties of the g3p protein oramyloid binding fragment of g3p (e.g., the g3p protein or amyloidbinding fragment retains the same charge, structure, polarity,hydrophobicity/hydrophilicity, and/or preserves functions such as theability to recognize, bind to, and/or reduce amyloid). Such conservativeamino acid modifications are based on the relative similarity of theamino acid side-chain substituents, for example, their hydrophobicity,hydrophilicity, charge, size, and the like. Exemplary sets of aminoacids which are interchangeable as conservative substitutions, and whichtake various of the foregoing characteristics into consideration, arewell known to those of skill in the art and include: arginine andlysine; glutamate and aspartate; serine and threonine; glutamine andasparagine; and valine, leucine, and isoleucine.

The term “immunogenic” or “immunogenicity” is used herein to refer tothe ability of a composition to elicit an immune response in a mammalthat has been exposed to the composition. In some aspects, the presentinvention relates to polypeptides or compositions with reducedimmunogenicity or that are fully deimmunized. Full deimmunizationindicates the removal of all five of the T-cell recognition epitopespresent in the native GAIM amino acid sequence by one or more mutationsin those epitopic sequences. Such deimmunizing mutations may constitutea substitution, insertion, or deletion of one or more amino acidresidues in an epitope, or may constitute partially or fully deletingthe epitopic sequence.

General Amyloid Interaction Motif (GAIM) of G3p and GAIM-Ig FusionsThereof

The general amyloid interaction motif (GAIM) is a two-domain polypeptidecomprising the N1 and N2 domains of g3p. The GAIM N2 domain consists ofthree distinct structural elements: a globular part resembling N1 instructure (Holliger et al. (1999) J Mol Biol, 288:649-57), analpha-helix, and a disordered region that forms an extensive network ofH-bonds with the N1 domain. The N2 hinge region also contains severalproline residues, one of which (P213) has been implicated in maintainingGAIM in an open, ToIA binding-competent state. The N1 and the N2 domainsof the GAIM monomer have an asymmetric distribution of aromatic aminoacids. The GAIM N2 domain contains 11 Tyrosine (Tyr) residues and 1exposed Tryptophan (Trp) residue; the N1 domain contains 3 Trp and 3 Tyrresidues. Thus, the intrinsic fluorescence of tyrosine and tryptophanresidues allow specific monitoring of conformational changes in N2 andN1 domains, respectively (Martin and Schmid (2003) J Mol Biol,405:989-1003), allowing for detection of an open conformation of GAIM.

H/D exchange studies show that GAIM binds to the central core of Aβ42fibers. As demonstrated by FIG. 2, H/D exchange studies also show thatGAIM engages discontinuous sequences on the fibril-core and binds bothsequences rich in aromatic residues (e.g., residues 17-25 in Aβ42) andaliphatic residues (e.g., residues 31-40 in Aβ42). This results inrobust inhibition of amyloid assembly and efficient remodeling of fibersinto amorphous aggregates (Krishnan et al. (2014) J Mol Biol,426:2500-19).

In some aspects, a polypeptide or composition comprising the polypeptidecomprises a GAIM variant. In some embodiments, a GAIM variant has nomore than 25 amino acid differences as compared to the referencesequence. In some embodiments, a GAIM variant has no more than 17 aminoacid differences as compared to the reference sequence. In someembodiments, a GAIM variant has no more than 10 amino acid differencesas compared to the reference sequence. In some embodiments, a varianthas no more than 7 amino acid differences as compared to the referencesequence. In some embodiments, the reference sequence is SEQ ID NO:12(GAIM portion of PB120). In some embodiments, the reference sequence isSEQ ID NO:14 (GAIM portion of PB106). In some embodiments, the referencesequence is SEQ ID NO:16 (GAIM portion of “PB106+EDGS” (“EDGS” disclosedas SEQ ID NO:54)).

Unless otherwise specified, all GAIM amino acid sequence numbering isbased on the amino acid sequence of SEQ ID NO:16 and all GAIM-Ig aminoacid sequence numbering is based on the amino acid sequence of SEQ IDNO:15, which constitutes SEQ ID NO:16 fused at the C-terminal end to ahuman IgG1-Fc amino acid sequence by the short linker ARS.

Polypeptides of the invention comprise any of the GAIM variantsdescribed herein. The GAIM variants disclosed herein comprise asubstitution of SEQ ID NO:3 (DDKTLD; amino acids 24-29 relative to SEQID NO:13) with SEQ ID NO:4 (EGDS). This substitution is present inreference sequence SEQ ID NO: 16 and results in the GAIM variants of theinvention having an open-conformation (“open” or “unlocked”) GAIMvariant.

The GAIM variants of the invention also include at least one additionalset of amino acid changes selected from (i) alternative T-cell epitope1-deimmunizing changes, (ii) T-cell epitope 2-deimmunizing changes, and(iii) N2-stabilizing changes.

In some embodiments, the at least one other set of amino acid changesincreases amyloid affinity while still being deimmunized in T-cellepitope 1. In reference SEQ ID NO:16, deimmunized T-cell epitope 1 spansfrom amino acids G47 to H55. The H55 in that sequence causes thedeimmunization; wild-type g3p (in which T-cell epitope 1 is notdeimmunized) has a threonine at the corresponding position. Althoughamyloid affinity remains significant for g3p polypeptide variantscomprising the threonine-to-histidine change at amino acid 55 of SEQ IDNO:16, this affinity is somewhat reduced relative to wild-type.Interestingly, it has been reported that that the sequence YGT, which ispresent in native g3p, is a ToIA binding motif (S Pommier et al, J.Bacteriol. (2005), 187 (21), pp. 7526-34). Without being bound bytheory, we believe that GAIM-amyloid binding may require similar aminoacid interactions as g3p-ToIA binding. Thus, we explored regeneratingthe 53YGT55 sequence in SEQ ID NO:16 by making a H55T substitution(e.g., reverting to the wild-type T-cell epitope 1 sequence) and lookingfor an alternative substitution in the now-regenerated T-cell epitopethat would affect deimmunization. We found that a T50 substitutioncauses deimmunization of T-cell epitope 1 without affecting amyloidaffinity. Therefore, in some embodiments, a GAIM variant of theinvention comprises a T50 substitution accompanied by a H55Tsubstitution. In some aspects of these embodiments, the T50 substitutionis T50R, T50K, T50G, or T50H. In at least one aspect of theseembodiments, the T50 substitution is T50H.

In some embodiments, the at least one other set of amino acid changesdeimmunizes T-cell epitope 2 without significantly altering amyloidaffinity. In reference SEQ ID NO:16, T-cell epitope 2 spans from aminoacid M134 to N142 (see U.S. Pat. No. 9,988,444 B2 and U.S. PatentPublication US 2018/0207231 A1, each incorporated by reference in itsentirety), and is unchanged as compared to the corresponding wild-typeg3p sequence. Prior to the present invention, we have been unable todeimmunize this epitope without significant reduction in amyloid bindingand/or significant decrease in the stability of the resulting GAIM. Wehave now discovered that substitution of N142 and/or N137 with anotheramino acid deimmunizes T-cell epitope 2 without significant effect onamyloid binding or stability. Thus, in some embodiments, a GAIM variantof the invention comprises a substitution of N142. In some aspects ofthese embodiments, the N142 substitution is N142A. In alternateembodiments, a GAIM variant of the invention comprises a substitution ofN137. In some aspects of these embodiments, the N137 substitution isN137G. In other alternate embodiments, a GAIM variant of the inventioncomprises a substitution of N137 and a substitution of N142. In someaspects of these embodiments, the N137 substitution is N137G and theN142 substitution is N142A.

In some embodiments, the at least one other set of amino acid changesincreases the stability of N2. These changes target one or more of theso-called slow folding loops present in SEQ ID NO:16, which span aminoacids 135-138 (FQNN; SEQ ID NO:5; Turn 1), 143-146 (RQGA; SEQ ID NO:6;Turn 2), and 156-162 (QGTDPVK; SEQ ID NO:7; Turn 3), as depicted by FIG.3C. We have discovered that certain amino acid substitutions and/ordeletions in one or more of these regions will increase the stability ofN2 in a GAIM. Thus, in some embodiments, at least one other set of aminoacid changes is selected from: (i) N137G; (ii) R143V, Q144N, and,optionally, A146V, A146T, or A146K; and (iii) V161G, deletion of T158,D159, and P160, optionally, Q156V or Q156Y, and, optionally, G157N. Asprovided above, the N137G substitution that stabilizes N2 by removingthe slow-folding loop at Turn 1 also deimmunizes T-cell epitope 2. Insome aspects of these embodiments, the GAIM variant comprises amino acidchanges in only one of Turn 1, Turn 2, and Turn 3, e.g., one of: (i)N137G; (ii) R143V, Q144N, and, optionally, A146V, A146T, or A146K; and(iii) V161G, deletion of T158, D159, and P160, optionally, Q156V orQ156Y, and, optionally, G157N. In some aspects of these embodiments, theGAIM variant comprises amino acid changes in at least two of the turns,e.g., two of (i) N137G; (ii) R143V, Q144N, and, optionally, A146V,A146T, or A146K; and (iii) V161G, deletion of T158, D159, and P160,optionally, Q156V or Q156Y, and, optionally, G157N. In some aspects ofthese embodiments, the amino acid change is N137G, resulting in SEQ IDNO:8 at amino acids 135-138. In some aspects of these embodiments, theamino acid change is R143V, Q144N, and A146V, resulting in SEQ ID NO:9at amino acids 143-146. In some aspects of these embodiments, the aminoacid change is deletion of T158, D159, and P160, and the substitutionV161G, resulting in SEQ ID NO:10 as a replacement for amino acids156-162. In at least one aspect of these embodiments, the GAIM variantdoes not comprise amino acid changes in all three of the turns.

In some embodiments, a polypeptide of the invention comprises a GAIMvariant having at least one set of amino acid changes selected from anyof the above-described alternative T-cell epitope 1-deimmunizing changesand at least one set of amino acid changes selected from any of theabove-described T-cell epitope 2-deimmunizing changes.

In some embodiments, the GAIM variants has at least one set of aminoacid changes selected from any of the above-described alternative T-cellepitope 1-deimmunizing changes and at least one set of amino acidchanges selected from any of the above-described N2-stabilizing changes.

In some embodiments, the GAIM variant has at least one set of amino acidchanges selected from any of the above-described T-cell epitope2-deimmunizing changes and at least one set of amino acid changesselected from any of the above-described N2-stabilizing changes.

In some embodiments, the GAIM variant has at least one set of amino acidchanges selected from any of the above-described alternative T-cellepitope 1-deimmunizing changes, at least one set of amino acid changesselected from any of the above-described T-cell epitope 2-deimmunizingchanges, and at least one set of amino acid changes selected from any ofthe above-described N2-stabilizing changes.

The choice of the specific set of amino acid changes for each of theaforementioned types of changes can be made from any of the changesdescribed herein for that given type. Non-limiting examples of sets ofamino acid changes can be found in Table 1.

TABLE 1 Mutations of Open-Stabilized GAIM-Ig Fusion Relative to SEQ IDNO:16 GAIM- T-cell T-cell N2- Glycosylation Ig SEQ Epitope 1 Epitope 2stabilizing Signal Fusion ID Muta- Muta- Muta- Muta- Protein NO:tion(s)* tion(s) tion(s) tion(s)*** PB108 29 None N137G** N137G NonePB122 30 None None R143V None Q144N A146V PB116 31 None N137G N137G NoneN142A PB114 32 None N142A R143V None Q144N A146V PB109 33 T50H N137GN137G None H55T N142A PB110 34 T50H N142A R143V None H55T Q144N A146VPB105 35 T50H N137G N137G None H55T PB127 36 T50H N137G N137G N38A H55T*T-cell epitope 1 of SEQ ID NO:16 is deimmunized by a T55H substitutionrelative to wild-type g3p. Mutations in T-cell epitope 1 in theopen-stabilized GAIM-Ig fusions represent alternative deimmunizingsubstitutions. **The N137G substitution deimmunizes T-cell epitope 2 andstabilizes the N2 domain. ***SEQ ID NO:16 is deglycosylated by a T40Gmutation relative to wild-type g3p. Mutations in the potentialglycosylation signal in the open-stabilized GAIM-Ig fusions representadditional deglycosylating substitutions.

The polypeptides of the invention comprise a deglycosylated GAIMvariant. The reference sequence SEQ ID NO:16 is deglycosylated becauseit comprises a T40G mutation relative to wild-type g3p, thus alteringthe native NAT glycosylation signal to NAG. Examples of otherdeglycosylated g3p mutants and/or variants can be found in U.S. PatentPublication US 2018/0207231 A1. However, GAIM variants that havedifferent and/or additional deglycosylating mutations in the nativeglycosylation signal are also part of the present invention. Forexample, the three amino acid sequence NX(T/S), where X is any aminoacid, is a known glycosylation signal. Substitution of the asparagine(N) in such a sequence with any amino acid other than cysteine willdestroy the glycosylation signal. Similarly, substitution of threonine(T) or serine (S) in such a sequence with any amino acid other thancysteine will destroy the glycosylation signal.

Thus, in some embodiments, a GAIM variant described herein comprises asubstitution of N38 with any amino acid other than cysteine as comparedto SEQ ID NO:16, and further comprises one or more of an alternativeT-cell epitope 1-deimmunizing change, a T-cell epitope 2-deimmunizingchange, and/or an N2-stabilizing change. In some aspects of theseembodiments, the substitution of N38 is N38A. In some aspects of theseembodiments, the GAIM variant further comprises a substitution of G40 toany amino acid other than cysteine.

In some alternate embodiments, a GAIM variant described herein comprisesa substitution of G40 with any amino acid other than cysteine,threonine, or serine as compared to SEQ ID NO:16, and further comprisesone or more of a T-cell epitope 1-deimmunizing change, a T-cell epitope2-deimmunizing change, and/or an N2-stabilizing change.

SEQ ID NO:16 includes the N-terminal amino acids M1 and A2. Recombinantproduction of GAIM in animal cell lines can result in polypeptides thatare missing M1 or both M1 and A2 (an “N-terminal truncation”). SuchN-terminal truncations do not affect amyloid-binding activity. Thus, insome embodiments, a GAIM variant optionally lacks amino acid 1 (ΔM1) orboth amino acids 1 and 2 (ΔM1 and ΔA2) of SEQ ID NO:16, in addition tocomprising one or more of a T-cell epitope 1-deimmunizing change, aT-cell epitope 2-deimmunizing change, and/or an N2-stabilizing change.As used herein, a GAIM variant lacking amino acid 1 or both amino acid 1or 2 may refer to an N-terminal truncation (i.e., removal aftertranslation) or a deletion mutation.

In some embodiments, the GAIM variant is a variant of SEQ ID NO:16comprising the substitution N137G. In some aspects of these embodiments,the GAIM variant lacks amino acid 1 (e.g., ΔM1). In some aspects ofthese embodiments, the GAIM variant lacks amino acids 1 and 2 (e.g., ΔM1and ΔA2). In some aspects of these embodiments, the GAIM variant furthercomprises a substitution of N38 with any amino acid other than cysteine,a substitution of G40 with any amino acid other than cysteine,threonine, or serine, or both a substitution of N38 with any amino acidother than cysteine and a substitution of G40 with any amino acid otherthan cysteine. In at least one aspect of these embodiments, the N38substitution is N38A.

In some embodiments, the GAIM variant is a variant of SEQ ID NO:16comprising R143V, Q144N, and, optionally, A146V, A146T, or A146K. Incertain embodiments, the GAIM variant is a variant of SEQ ID NO:16comprising R143V, Q144N, and A146V. In some aspects of theseembodiments, the GAIM variant additionally lacks amino acid 1 (e.g.,ΔM1). In some aspects of these embodiments, the GAIM variantadditionally lacks amino acids 1 and 2 (e.g., ΔM1 and ΔA2). In someaspects of these embodiments, the GAIM variant further comprises asubstitution of N38 with any amino acid other than cysteine, asubstitution of G40 with any amino acid other than cysteine, threonine,or serine, or both a substitution of N38 with any amino acid other thancysteine and a substitution of G40 with any amino acid other thancysteine. In at least one aspect of these embodiments, the N38substitution is N38A.

In some embodiments, the GAIM variant is a variant of SEQ ID NO:16comprising a substitution of T50 with any other amino acid, thesubstitution H55T, and the substitution N137G. In at least one aspect ofthese embodiments, the substitution of T50 is selected from T50H, T50G,T50K, and T50R. In at least one aspect of these embodiments, thesubstitution of T50 is T50H. In some aspects of these embodiments, theGAIM variant further (i) comprises an N142A substitution; (ii) comprisesa deglycosylating mutation of N38 and/or G40; (iii) lacks amino acid 1or both amino acids 1 and 2; or (iv) any combination thereof. Forexample, in some aspects of these embodiments, the GAIM variant lacksamino acid 1 (e.g., ΔM1). In some aspects of these embodiments, the GAIMvariant lacks amino acids 1 and 2 (e.g., ΔM1 and ΔA2). In some aspectsof these embodiments, the GAIM variant further comprises a substitutionof N38 with any amino acid other than cysteine, a substitution of G40with any amino acid other than cysteine, threonine, or serine, or both asubstitution of N38 with any amino acid other than cysteine and asubstitution of G40 with any amino acid other than cysteine. In at leastone aspect of these embodiments, the N38 substitution is N38A.

In certain embodiments, the GAIM variant is a variant of SEQ ID NO:16comprising the substitutions N137G and N142A. In some aspects of theseembodiments, the GAIM variant further (i) comprises a substitution ofT50 with any other amino acid as well as the substitution H55T; (ii)comprises a deglycosylating mutation of N38 and/or G40; (iii) lacksamino acid 1 or both amino acids 1 and 2; or (iv) any combinationthereof. In certain aspects of these embodiments, the substitution ofT50 is selected from T50H, T50G, T50K, and T50R. In at least one aspectof these embodiments, the substitution of T50 is T50H. In some aspectsof these embodiments, the GAIM variant lacks amino acid 1 (e.g., ΔM1).In some aspects of these embodiments, the GAIM variant lacks amino acids1 and 2 (e.g., ΔM1 and ΔA2). In some aspects of these embodiments, theGAIM variant further comprises a substitution of N38 with any amino acidother than cysteine, a substitution of G40 with any amino acid otherthan cysteine, threonine, or serine, or both a substitution of N38 withany amino acid other than cysteine and a substitution of G40 with anyamino acid other than cysteine. In at least one aspect of theseembodiments, the N38 substitution is N38A.

In certain embodiments, the GAIM variant is a variant of SEQ ID NO:16comprising the following substitutions: N142A, R143V, Q144N, and,optionally, A146V, A146T, or A146K. In certain embodiments, the GAIMvariant is a variant of SEQ ID NO:16 comprising the followingsubstitutions: N142A, R143V, Q144N, and A146V. In some aspects of theseembodiments, the GAIM variant further (i) comprises a substitution ofT50 with any other amino acid as well as the substitution H55T; (ii)comprises a deglycosylating mutation of N38 and/or G40; (iii) lacksamino acid 1 or both amino acids 1 and 2; or (iv) any combinationthereof. In some aspects of these embodiments, the substitution of T50is selected from T50H, T50G, T50K, and T50R. In at least one aspect ofthese embodiments, the substitution of T50 is T50H. In some aspects ofthese embodiments, the GAIM variant lacks amino acid 1 (ΔM1). In someaspects of these embodiments, the GAIM variant lacks amino acids 1 and 2(ΔM1 and ΔA2). In some aspects of these embodiments, the GAIM variantfurther comprises a substitution of N38 with any amino acid other thancysteine, a substitution of G40 with any amino acid other than cysteine,threonine, or serine, or both a substitution of N38 with any amino acidother than cysteine and a substitution of G40 with any amino acid otherthan cysteine. In at least one aspect of these embodiments, the N38substitution is N38A.

In certain embodiments, the GAIM variant is a variant of SEQ ID NO:16comprising the following substitutions: ΔT158, ΔD159, ΔP160, V161G and,optionally, (i) Q156V or Q156Y and/or (ii) G157N. In certain aspects ofthese embodiments, the GAIM variant is a variant of SEQ ID NO:16comprising the following substitutions: ΔT158, ΔD159, ΔP160, and V161G.In some aspects of these embodiments, the GAIM variant further (i)comprises a substitution of T50 with any other amino acid as well as thesubstitution H55T; (ii) comprises a deglycosylating mutation of N38and/or G40; (iii) lacks amino acid 1 or both amino acids 1 and 2; or(iv) any combination thereof. In some aspects of these embodiments, thesubstitution of T50 is selected from T50H, T50G, T50K, and T50R. In atleast one aspect of these embodiments, the substitution of T50 is T50H.In some aspects of these embodiments, the GAIM variant lacks amino acid1 (ΔM1). In some aspects of these embodiments, the GAIM variant lacksamino acids 1 and 2 (ΔM1 and ΔA2). In some aspects of these embodiments,the GAIM variant further comprises a substitution of N38 with any aminoacid other than cysteine, a substitution of G40 with any amino acidother than cysteine, threonine, or serine, or both a substitution of N38with any amino acid other than cysteine and a substitution of G40 withany amino acid other than cysteine. In at least one aspect of theseembodiments, the N38 substitution is N38A.

In at least one embodiment, a polypeptide of the present inventioncomprises a GAIM variant having the amino acid sequence of SEQ ID NO:19.In at least one embodiment, a polypeptide of the present inventioncomprises a GAIM variant having the amino acid sequence of SEQ ID NO:20.In at least one embodiment, a polypeptide of the present inventioncomprises a GAIM variant having the amino acid sequence of SEQ ID NO:21.In at least one embodiment, a polypeptide of the present inventioncomprises a GAIM variant having the amino acid sequence of SEQ ID NO:22.In at least one embodiment, a polypeptide of the present inventioncomprises a GAIM variant having the amino acid sequence of SEQ ID NO:23.In at least one embodiment, a polypeptide of the present inventioncomprises a GAIM variant having the amino acid sequence of SEQ ID NO:24.In at least one embodiment, a polypeptide of the present inventioncomprises a GAIM variant having the amino acid sequence of SEQ ID NO:25.In at least one embodiment, a polypeptide of the present inventioncomprises a GAIM variant having the amino acid sequence of SEQ ID NO:26.

Any of the above-described GAIM variants may be fused at the C-terminalend, directly or through a short linker, to an immunoglobulin constantregion, to yield a GAIM-Ig fusion protein. The immunoglobulin constantregion of the GAIM-Ig fusion proteins described herein may be theimmunoglobulin constant region of IgG (including IgG1, IgG2, IgG3, andIgG4), IgA, IgD, IgE, or IgM. In some aspects, the immunoglobulinconstant region is IgG. In certain aspects, the IgG is IgG1. In otheraspects, the IgG is IgG2. In some embodiments, the immunoglobulinconstant region is a human immunoglobulin constant region. In someembodiments, the immunoglobulin constant region is the Fc portion of ahuman IgG, or a fragment thereof. Fc portions of a human IgG suitablefor the fusion proteins of the invention include wild-type or modifiedFc portions. For example, a suitable modified Fc portion of a human IgGmay stabilize the fusion protein and/or increase its half-life relativeto wild-type Fc. Nonlimiting examples of modified Fc include thosedisclosed in U.S. Pat. Nos. 7,083,784, 7,217,797, 7,217,798, U.S. patentapplication Ser. No. 14/214,146, and WO-1997034631. In at least oneembodiment, the immunoglobulin constant region is the Fc portion ofhuman IgG1. In at least one embodiment, the immunoglobulin constantregion is the Fc portion of human IgG2. In some embodiments, theimmunoglobulin constant region of the GAIM-Ig fusion protein comprises aC-terminal lysine (e.g., K485). In other embodiments, the GAIM-Ig fusionlacks a C-terminal lysine (e.g., ΔK485).

In some embodiments, the GAIM-Ig fusion protein consists essentially ofa polypeptide comprising any GAIM variant disclosed herein and the Fcportion of a human IgG (e.g., human IgG1). In at least one embodiment,the GAIM-Ig fusion protein consists essentially of a sequence at least95%, 96%, 97%, 98%, or 99% identical to that described by SEQ ID NO:19and the Fc portion of a human IgG (e.g., human IgG1). In some aspects ofthis embodiment, the amino acid sequence of the GAIM portion of theGAIM-Ig fusion protein differs from that described by SEQ ID NO: 19 by10-15, 1-10, or 1-5 conservative substitutions. In other aspects of thisembodiment, the GAIM-Ig fusion protein consists essentially of SEQ IDNO:19 and the Fc portion of a human IgG (e.g., human IgG1). For example,in some aspects, the GAIM-Ig fusion protein consists essentially of theamino acid sequence of SEQ ID NO:29 (PB108). In other aspects of thisembodiment, the GAIM-Ig fusion protein consists essentially of a variantof SEQ ID NO:29 in which the variant lacks amino acid 1 (ΔM1), aminoacids 1 and 2 (ΔM1 and ΔA2), amino acid 485 (ΔK485), amino acids 1 and485 (ΔM1 and ΔK485), or amino acids 1, 2, and 485 (ΔM1, ΔA2, and ΔK485).

In at least one embodiment, the GAIM-Ig fusion protein consistsessentially of a sequence at least 95%, 96%, 97%, 98%, or 99% identicalto that described by SEQ ID NO:20 and the Fc portion of a human IgG(e.g., human IgG1). In some aspects of this embodiment, the amino acidsequence of the GAIM portion of the GAIM-Ig fusion protein differs fromthat described by SEQ ID NO: 20 by 10-15, 1-10, or 1-5 conservativesubstitutions. In other aspects of this embodiment, the GAIM-Ig fusionprotein consists essentially of SEQ ID NO:20 and the Fc portion of ahuman IgG (e.g., human IgG1). For example, in some aspects, the GAIM-Igfusion protein consists essentially of the amino acid sequence of SEQ IDNO:30 (PB122). In other aspects of this embodiment, the GAIM-Ig fusionprotein consists essentially of a variant of SEQ ID NO:30 in which thevariant lacks amino acid 1 (ΔM1), amino acids 1 and 2 (ΔM1 and ΔA2),amino acid 485 (ΔK485), amino acids 1 and 485 (ΔM1 and ΔK485), or aminoacids 1, 2, and 485 (ΔM1, ΔA2, and ΔK485).

In at least one embodiment, the GAIM-Ig fusion protein consistsessentially of a sequence at least 95%, 96%, 97%, 98%, or 99% identicalto that described by SEQ ID NO:21 and the Fc portion of a human IgG(e.g., human IgG1). In some aspects of this embodiment, the amino acidsequence of the GAIM portion of the GAIM-Ig fusion protein differs fromthat described by SEQ ID NO: 21 by 10-15, 1-10, or 1-5 conservativesubstitutions. In other aspects of this embodiment, the GAIM-Ig fusionprotein consists essentially of SEQ ID NO:21 and the Fc portion of ahuman IgG (e.g., human IgG1). For example, in some aspects, the GAIM-Igfusion protein consists essentially of the amino acid sequence of SEQ IDNO:31 (PB116). In other aspects of this embodiment, the GAIM-Ig fusionprotein consists essentially of a variant of SEQ ID NO:31 in which thevariant lacks amino acid 1 (ΔM1), amino acids 1 and 2 (ΔM1 and ΔA2),amino acid 485 (ΔK485), amino acids 1 and 485 (ΔM1 and ΔK485), or aminoacids 1, 2, and 485 (ΔM1, ΔA2, and ΔK485).

In at least one embodiment, the GAIM-Ig fusion protein consistsessentially of a sequence at least 95%, 96%, 97%, 98%, or 99% identicalto that described by SEQ ID NO:22 and the Fc portion of a human IgG(e.g., human IgG1). In some aspects of this embodiment, the amino acidsequence of the GAIM portion of the GAIM-Ig fusion protein differs fromthat described by SEQ ID NO: 22 by 10-15, 1-10, or 1-5 conservativesubstitutions. In other aspects of this embodiment, the GAIM-Ig fusionprotein consists essentially of SEQ ID NO:22 and the Fc portion of ahuman IgG (e.g., human IgG1). For example, in some aspects, the GAIM-Igfusion protein consists essentially of the amino acid sequence of SEQ IDNO:32 (PB114). In other aspects of this embodiment, the GAIM-Ig fusionprotein consists essentially of a variant of SEQ ID NO:32 in which thevariant lacks amino acid 1 (ΔM1), amino acids 1 and 2 (ΔM1 and ΔA2),amino acid 485 (ΔK485), amino acids 1 and 485 (ΔM1 and ΔK485), or aminoacids 1, 2, and 485 (ΔM1, ΔA2, and ΔK485).

In at least one embodiment, the GAIM-Ig fusion protein consistsessentially of a sequence at least 95%, 96%, 97%, 98%, or 99% identicalto that described by SEQ ID NO:23 and the Fc portion of a human IgG(e.g., human IgG1). In some aspects of this embodiment, the amino acidsequence of the GAIM portion of the GAIM-Ig fusion protein differs fromthat described by SEQ ID NO: 23 by 10-15, 1-10, or 1-5 conservativesubstitutions. In other aspects of this embodiment, the GAIM-Ig fusionprotein consists essentially of SEQ ID NO:23 and the Fc portion of ahuman IgG (e.g., human IgG1). For example, in some aspects, the GAIM-Igfusion protein consists essentially of the amino acid sequence of SEQ IDNO:33 (PB109). In other aspects of this embodiment, the GAIM-Ig fusionprotein consists essentially of a variant of SEQ ID NO:33 in which thevariant lacks amino acid 1 (ΔM1), amino acids 1 and 2 (ΔM1 and ΔA2),amino acid 485 (ΔK485), amino acids 1 and 485 (ΔM1 and ΔK485), or aminoacids 1, 2, and 485 (ΔM1, ΔA2, and ΔK485).

In at least one embodiment, the GAIM-Ig fusion protein consistsessentially of a sequence at least 95%, 96%, 97%, 98%, or 99% identicalto that described by SEQ ID NO:24 and the Fc portion of a human IgG(e.g., human IgG1). In some aspects of this embodiment, the amino acidsequence of the GAIM portion of the GAIM-Ig fusion protein differs fromthat described by SEQ ID NO: 24 by 10-15, 1-10, or 1-5 conservativesubstitutions. In other aspects of this embodiment, the GAIM-Ig fusionprotein consists essentially of SEQ ID NO:24 and the Fc portion of ahuman IgG (e.g., human IgG1). For example, in some aspects, the GAIM-Igfusion protein consists essentially of the amino acid sequence of SEQ IDNO:34 (PB110). In some embodiments, the GAIM-Ig fusion protein consistsessentially of a variant of SEQ ID NO:34 in which the variant lacksamino acid 1 (ΔM1), amino acids 1 and 2 (ΔM1 and ΔA2), amino acid 485(ΔK485), amino acids 1 and 485 (ΔM1 and ΔK485), or amino acids 1, 2, and485 (ΔM1, ΔA2, and ΔK485).

In at least one embodiment, the GAIM-Ig fusion protein consistsessentially of a sequence at least 95%, 96%, 97%, 98%, or 99% identicalto that described by SEQ ID NO:25 and the Fc portion of a human IgG(e.g., human IgG1). In some aspects of this embodiment, the amino acidsequence of the GAIM portion of the GAIM-Ig fusion protein differs fromthat described by SEQ ID NO: 25 by 10-15, 1-10, or 1-5 conservativesubstitutions. In other aspects of this embodiment, the GAIM-Ig fusionprotein consists essentially of SEQ ID NO:25 and the Fc portion of ahuman IgG (e.g., human IgG1). For example, in some aspects, the GAIM-Igfusion protein consists essentially of the amino acid sequence of SEQ IDNO:35 (PB105). In other aspects of this embodiment, the GAIM-Ig fusionprotein consists essentially a variant of SEQ ID NO:35 in which thevariant lacks amino acid 1 (ΔM1), amino acids 1 and 2 (ΔM1 and ΔA2),amino acid 485 (ΔK485), amino acids 1 and 485 (ΔM1 and ΔK485), or aminoacids 1, 2, and 485 (ΔM1, ΔA2, and ΔK485).

In at least one embodiment, the GAIM-Ig fusion protein consistsessentially of a sequence at least 95%, 96%, 97%, 98%, or 99% identicalto that described by SEQ ID NO:26 and the Fc portion of a human IgG(e.g., human IgG1). In some aspects of this embodiment, the amino acidsequence of the GAIM portion of the GAIM-Ig fusion protein differs fromthat described by SEQ ID NO: 26 by 10-15, 1-10, or 1-5 conservativesubstitutions. In other aspects of this embodiment, the GAIM-Ig fusionprotein consists essentially of SEQ ID NO:26 and the Fc portion of ahuman IgG (e.g., human IgG1). For example, in some aspects, the GAIM-Igfusion protein consists essentially of the amino acid sequence of SEQ IDNO:36 (PB127). In other aspects of this embodiment, the GAIM-Ig fusionprotein consists essentially of a variant of SEQ ID NO:36 in which thevariant lacks amino acid 1 (ΔM1), amino acids 1 and 2 (ΔM1 and ΔA2),amino acid 485 (ΔK485), amino acids 1 and 485 (ΔM1 and ΔK485), or aminoacids 1, 2, and 485 (ΔM1, ΔA2, and ΔK485).

In some aspects of the invention, the GAIM portion and Ig portion of theGAIM-Ig fusions described herein are connected by a small linker. Insome embodiments, the small linker is rich in glycine, serine, and/orthreonine, comprising at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% glycine, serine, and/or threonine. In someembodiments, the small linker comprises at least or about 50%, 55%, 60%,70%, or 75% glycine, serine, and/or threonine. In some embodiments, thesmall linker is comprised substantially or entirely of glycine, serine,and/or threonine. A small linker of a GAIM-Ig fusion may be up to 25amino acids in length, for example, from 1 to 5 amino acids in length,from 1 to 20 amino acids in length, from 5 to 10 amino acids in length,from 5 to 25 amino acids in length, or from 10 to 25 amino acids inlength. Small linkers may have, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 aminoacids. In some embodiments, the small linker does not contain a humanT-cell epitope or create a human T-cell epitope with either the GAIMvariant or Fc domain to which it is bound. Exemplary small linkersinclude linkers having various numbers of repeats of the sequence GGGGS(4GS; SEQ ID NO:27) or GGGS (3GS; SEQ ID NO:28), such as from 2, 3, 4,to 5 repeats of such a sequence. Exemplary small linkers may include oneor more lysine residues. Other exemplary small linkers include the aminoacid sequence ARS.

The GAIM-Ig fusions described herein demonstrate several advantages overthe prior art. Studies aimed at identifying the pathological forms of Aβin the Alzheimer's disease (ΔD)-brain have shown that both insolubleplaque and soluble Aβ consists of a heterogeneous population of N- andC-terminal truncated Aβ peptides (Wildburger et al., (2017) Sci Rep7:9520) forming structurally diverse conformations (Condello et al.(2018) PNAS, 115:E782-91; Rasmussen et al. (2017) PNAS, 114:13018-23;Liu et al. (2016) Sci Rep, 6:33079). It is also observed that majorityof N-terminally truncated Aβ fragments constitute the major part of theamyloid plaque (Wildburger et al., (2017) Sci Rep 7:9520). However, themajority of antibody-related therapies against amyloid aggregation ormisfolding have failed in the clinic at least in part due to theirinability to effectively engage with N-terminally truncated or modifiedforms of amyloid. The GAIM-Ig fusions of the invention address thepreviously unmet need for a composition that can target a variety ofamyloid proteins, as these fusions are capable of engaging various Aβaggregates, even aggregates having different morphologies andaggregation properties.

The GAIM-Ig fusions disclosed herein bind, among other aggregates, thetruncated 11-42 Aβ aggregates and/or post-translationally-modifiedpyro-glutamate Aβ aggregates, both of which are clinically relevant toAlzheimer's disease. As shown further in Tables 2 and 3, the GAIM-Igfusions of the invention target multiple types of amyloid protein,including but not limited to Aβ aggregates, N-terminal truncated Aβaggregates, tau, multiple conformers of transthyretin (TTR), and diversemorphologies of immunoglobulin light chain (LC) aggregates. Thesetargets include amyloid protein found in patients at risk of orsuffering from diseases described herein.

Antibody-related therapies of the prior art may also fail in the clinicbecause they fail to block aggregation of phosphorylated tau and/or failto block tau's spread from one region of the brain to another. Theopen-stabilized GAIM-Ig fusions of the present invention address thisneed. The GAIM-Ig fusions of the invention cause remodeling of tau, thuspreventing tau aggregates from seeding soluble tau and blocking tauaggregate propagation.

Taken together, GAIM-Ig fusions disclosed herein present a uniqueapproach to prevent or remove pathological amyloid aggregates. Relativeto prior art alternatives, the open-stabilized GAIM fusions describedherein show greater potency, structural stability, and specificity toamyloid, including both Aβ and tau fibers, and are also either partiallyor fully deimmunized.

Preparation of Polypeptides

Polypeptides of the invention (e.g., polypeptides comprising one or moreGAIM variants, including fusion proteins) can be synthesized usingtechniques well known in the art. For example, the polypeptides of theinvention can be synthesized recombinantly in cells (see, e.g., Sambrooket al. 1989, Molecular Cloning A Laboratory Manual, Cold Spring HarborLaboratory, N.Y. and Ausubel et al. 1989, Current Protocols in MolecularBiology, Greene Publishing Associates and Wiley Interscience, N.Y.).Alternatively, the polypeptides of the invention can be synthesizedusing known synthetic methods such as solid phase synthesis. Synthetictechniques are well known in the art (see, e.g., Merrifield, 1973,Chemical Polypeptides, (Katsoyannis and Panayotis eds.) pp. 335-61;Merrifield 1963, J. Am. Chem. Soc. 85:2149; Davis et al. 1985, Biochem.Intl. 10:394; Finn et al. 1976, The Proteins (3d ed.) 2:105; Erikson etal. 1976, The Proteins (3d ed.) 2:257; U.S. Pat. No. 3,941,763.Alternatively, the final construct may share essentially the samefunction as a recombinantly produced fusion protein, but simply beproduced using non-recombinant techniques, such as ligation chemistry.Components of the fusion proteins may be prepared using the same generalmethodology described for g3p expression and g3p mutations.

In some embodiments, the polypeptide may be fused to a marker sequence,such as a peptide that facilitates purification of the fused polypeptide(either alone or in addition to fusion to another protein orincorporation of a carrier molecule). The marker amino acid sequence maybe a hexa-histidine peptide (SEQ ID NO:53) such as the tag provided in apQE vector (Qiagen, Mississauga, Ontario, Canada), among others, many ofwhich are commercially available. As described in Gentz et al., Proc.Natl. Acad. Sci. (1989) 86:821-824, for instance, hexa-histidine (SEQ IDNO:53) provides for convenient purification of the fusion protein.Another peptide tag useful for purification, the hemagglutinin (HA) tag,corresponds to an epitope derived from the influenza HA protein. (Wilsonet al., (1984) Cell 37:767).

Pharmaceutical Compositions

In some embodiments, the invention provides a pharmaceutical compositioncomprising any polypeptide comprising a GAIM variant described herein,optionally together with a pharmaceutically acceptable carrier, diluentor excipient. A “pharmaceutical composition” refers to a therapeuticallyeffective amount of a composition as described herein with aphysiologically suitable carrier and/or excipient. A pharmaceuticalcomposition does not cause significant irritation to an organism. Thephrases “physiologically suitable carrier” and “pharmaceuticallyacceptable carrier” may be used interchangeably to refer to a carrier ora diluent that does not cause significant irritation to an organism anddoes not abrogate the biological activity and properties of theadministered composition. The term “excipient” refers to an inertsubstance added to a pharmaceutical composition to further facilitateadministration of an active ingredient. Examples, without limitation,include, saline, calcium carbonate, calcium phosphate, various sugarsand types of starch, cellulose derivatives, gelatin, vegetable oils,polyethylene glycols, and surfactants, including, for example,polysorbate 20.

Pharmaceutical compositions for use in accordance with the presentinvention may be formulated in a conventional manner using one or morephysiologically acceptable carriers comprising excipients andauxiliaries, which facilitate processing of the active ingredients intocompositions which can be used pharmaceutically. Proper formulation isdependent upon the route of administration chosen and upon the nature ofthe composition delivered (e.g., size and solubility of thepolypeptide). In one aspect of these embodiments, the pharmaceuticalcomposition is formulated for injection or infusion into the bloodstreamof a patient. In another aspect of these embodiments, the pharmaceuticalcomposition is formulated for direct administration to the brain orcentral nervous system of the patient, for example, by directintramedullary, intrathecal, or intraventricular injection.

The compositions described herein may be formulated for parenteraladministration, e.g., by bolus injection or continuous infusion.Pharmaceutical compositions for parenteral administration includeaqueous solutions of the composition in water-soluble form.Additionally, suspensions of the active ingredients may be prepared asoily or water-based injection suspensions. Suitable lipophilic solventsor vehicles include fatty oils such as sesame oil, or synthetic fattyacids esters such as ethyl oleate, triglycerides or liposomes. Aqueousinjection suspensions may contain substances, which increase theviscosity of the suspension, such as sodium carboxymethyl cellulose,sorbitol or dextran. Optionally, the suspension may also containsuitable stabilizers or agents (e.g., surfactants such as polysorbate(Tween 20)) which increase the solubility of the active ingredients toallow for the preparation of highly-concentrated solutions. Aprotein-based agent such as, for example, albumin may be used to preventadsorption of polypeptide of the invention to the delivery surface(i.e., IV bag, catheter, needle, etc.).

For oral administration, the pharmaceutical compositions can beformulated by combining the polypeptides described herein withpharmaceutically acceptable carriers well known in the art.

Formulations may be presented in unit dosage form, e.g., in vials,ampoules or in multidose containers with optionally, an addedpreservative. The compositions may be suspensions, solutions oremulsions in oily or aqueous vehicles, and may contain formulatoryagents such as suspending, stabilizing and/or dispersing agents. Singledosage forms may be in a liquid or a solid form. Single dosage forms maybe administered directly to a patient without modification or may bediluted or reconstituted prior to administration. In certainembodiments, a single dosage form may be administered in bolus form,e.g., single injection, single oral dose, including an oral dose thatcomprises multiple tablets, capsule, pills, etc. In alternateembodiments, a single dosage form may be administered over a period oftime, such as by infusion, or via an implanted pump, such as an ICVpump. In the latter embodiment, the single dosage form may be aninfusion bag or pump reservoir pre-filled with the appropriate amount ofa polypeptide comprising a GAIM variant. Alternatively, the infusion bagor pump reservoir may be prepared just prior to administration to apatient by mixing an appropriate dose of the polypeptide comprising aGAIM variant with the infusion bag or pump reservoir solution.

Another aspect of the invention includes methods for preparing apharmaceutical composition of the invention. Techniques for formulationof drugs may be found, for example, in “Remington's PharmaceuticalSciences,” Mack Publishing Co., Easton, Pa., latest edition, which isincorporated herein by reference in its entirety.

Pharmaceutical compositions suitable for use in the context of thepresent invention include compositions wherein the active ingredientsare contained in an amount effective to achieve the intended purpose.

Determination of a therapeutically or diagnostically effective amount iswell within the capability of those skilled in the art, especially inlight of the detailed disclosure provided herein.

Dosage amount and interval may be adjusted individually to provide brainlevels of the phage display vehicle which are sufficient to treat ordiagnose a particular brain disease, disorder, or condition (minimaleffective concentration, MEC). The MEC will vary for each preparationbut can be estimated from in vitro data. Dosages necessary to achievethe MEC will depend on individual characteristics.

Dosage intervals can also be determined using the MEC value.Preparations should be administered using a regimen that maintains brainlevels above the MEC for 10-90% of the time, preferably between 30-90%of the time and most preferably 50-90% of the time.

Depending on the severity and responsiveness of the disease to betreated, dosing can be of a single or a plurality of administrations,with a course of treatment lasting from several days to several weeks oruntil cure is effected or diminution of the disease state is achieved.

The amount of a composition to be administered will, of course, bedependent on the subject being treated or diagnosed, the severity of theaffliction, the judgment of the prescribing physician, etc.

Compositions of the present invention may, if desired, be presented in apack or dispenser device, such as an FDA approved kit, which may containone or more unit dosage forms containing the active ingredient. The packmay, for example, comprise metal or plastic foil, such as a blisterpack. The pack or dispenser device may be accompanied by instructionsfor administration. The pack or dispenser may also be accommodated by anotice associated with the container in a form prescribed by agovernmental agency regulating the manufacture, use or sale ofpharmaceuticals, which notice is reflective of approval by the agency ofthe form of the compositions or human or veterinary administration. Suchnotice, for example, may be of labelling approved by the U.S. Food andDrug Administration for prescription drugs or of an approved productinsert. Compositions comprising a preparation of the inventionformulated in a compatible pharmaceutical carrier may also be prepared,placed in an appropriate container, and labelled for treatment of anindicated disease, as further detailed herein.

Therapeutic Uses

Another aspect of the invention relates to the use of any of thepolypeptides, nucleic acid molecules, or compositions of the invention,in the treatment one or more diseases associated with misfolded and/oraggregated amyloid protein, including, but not limited to, thosediseases involving any of: transthyretin, immunoglobulin light chain(kappa or lambda), fAβ42, fasyn, fNM, or ftau.

In the context of treatments, the terms “patient”, “subject,” and“recipient” are used interchangeably and include humans as well as othermammals. In some embodiments, a patient is a human who is positive for abiomarker associated with a protein misfolding disease. In oneembodiment, the patient exhibits β-amyloid deposits as detected by PETimaging with florbetapir.

The term “treating” and its cognates refer to reducing, slowing, orreversing the progression of a disease in a patient exhibiting one ormore clinical symptoms of a disease. “Treating” also refers to reducing,slowing, or reversing the symptoms of a disease in a patient exhibitingone more clinical symptoms of a disease. In one embodiment, the patientexhibits β-amyloid deposits as detected by PET imaging with florbetapirand the number of β-amyloid deposits is reduced by the treatment. In oneembodiment, the patient exhibits β-amyloid deposits as detected by thepolypeptide or polypeptide compositions of the present invention and thenumber of β-amyloid deposits are reduced or maintained by the treatment.In another embodiment, the patient exhibits any type of amyloid depositsas detected by PET imaging and the cognitive function of the patient isimproved by the treatment. Improvement in cognitive function may beassayed by the methods and tests of McKhann et al., Alzheimer's &Dementia 7(3):263-9(2011).

“Prophylaxis” or “prevention” (used herein interchangeably) is distinctfrom treating and refers to administration of a polypeptide, nucleicacid, or composition to an individual before the onset of any clinicalsymptoms. Prophylaxis using any of the polypeptides, nucleic acids, orcompositions thereof of the present invention is encompassed.Prophylaxis may be implicated in individuals who are known to be atincreased risk for a disease, or who are certain to develop a disease,solely on the basis of one or more genetic markers. Many genetic markershave been identified for the various protein misfolding diseases. Forexample, individuals with one or more of the Swedish mutation, Indianamutation, or London mutation in hAPP are at increased risk fordeveloping early-onset Alzheimer's Disease and so are candidates forprophylaxis. Likewise, individuals with the trinucleotide CAG repeats inthe huntingtin gene, particularly those with 36 or more repeats, willeventually develop Huntington's Disease and so are candidates forprophylaxis.

Diseases associated with or characterized by misfolded and/or aggregatedamyloid protein encompass diseases associated with (e.g., caused orcorrelated at least in part by) misfolded amyloid protein, aggregatedamyloid protein, or both misfolded and aggregated amyloid protein.Peptides or proteins that may form amyloid are described above. Forexample, in some embodiments, amyloid is formed by Aβ, including but notlimited to Aβ40, Aβ42, N-truncated Aβ11-42, Aβ11-42-Pyro, Aβ3-42-Pyro,Aβ1-42-E22Q-Dutch mutation, or a combination thereof. In someembodiments, amyloid is formed a prion protein, e.g., PrP^(sc). In someembodiments, amyloid is formed by transthyretin. In some embodiments,amyloid is formed by immunoglobulin light chain, for exampleimmunoglobulin kappa light chain and/or immunoglobulin lambda lightchain. In some embodiments, amyloid is formed by tau. In someembodiments, amyloid is formed by α-synuclein.

Diseases associated with or characterized by misfolded and/or aggregatedamyloid protein are described above. Many of the above misfolded and/oraggregated amyloid protein diseases occur in the central nervous system(CNS). Nonlimiting examples of diseases occurring in the CNS areParkinson's Disease; Alzheimer's Disease; frontotemporal dementia (FTD)including those patients having the following clinical syndromes:behavioral variant FTD (bvFTD), progressive non-fluent aphasia (PNFA)and semantic dementia (SD); frontotemporal lobar degenerations (FTLDs);and Huntington's Disease. The polypeptides, nucleic acids, andcompositions of the invention may be used to treat diseasescharacterized by misfolded and/or aggregated amyloid protein that occurin the central nervous system (CNS).

Misfolding and/or aggregation of proteins may also occur outside theCNS. Amyloidosis A (AA) (for which the precursor protein is serum acutephase apolipoprotein, SAA) and multiple myeloma (precursor proteinsimmunoglobulin light and/or heavy chain) are two widely known proteinmisfolding and/or aggregated protein diseases that occur outside theCNS. Other examples include disease involving amyloid formed byα2-microglobulin, transthyretin (e.g., FAP, FAC, SSA), (apo)serum AA,apolipoproteins AI, AII, and AIV, gelsolin (e.g., Finnish form of FAP),immunoglobulin light chain (kappa or lambda), lysozyme, fibrinogen,cystatin C (e.g., Cerebral Amyloid Angiopathy, Hereditary CerebralHemorrhage with Amyloidosis, Icelandic Type), calcitonin, procalcitonin,islet amyloid polypeptide (e.g., IAPP amyloidosis), atrial natriureticfactor, prolactin, insulin, lactahedrin, kerato-epithelin, lactoferrin,odontogenic ameloblast-associated protein, and semenogelin I. Thepolypeptides, nucleic acids, and compositions of the invention may beused to treat diseases involving misfolding and/or aggregation ofproteins occurring outside the CNS.

Diseases associated with or characterized by misfolded and/or aggregatedamyloid protein may also involve tau lesions. Reviewed in Lee et al.,Annu. Rev. Neurosci. 24:1121-159 (2001). Tau proteins aremicrotubule-associated proteins expressed in axons of both central andperipheral nervous system neurons. Neurodegenerative tauopathies(sometimes referred to as tauopathies) are encompassed. Examples oftauopathies include Alzheimer's Disease, Amyotrophic lateralsclerosis/parkinsonism-dementia complex, Argyrophilic grain dementia,Corticobasal degeneration, Creutzfeldt-Jakob disease, Dementiapugilistica, diffuse neurofibrillary tangles with calcification, Down'ssyndrome, Frontotemporal dementias including frontotemporal dementiawith parkinsonism linked to chromosome 17,Gerstmann-Sträussler-Scheinker disease, Hallervorden-Spatz disease,Myotonic dystrophy, Niemann-Pick disease type C, Non-Guamanian motorneuron disease with neurofibrillary tangles, Pick's disease,Postencephalitic parkinsonism, Prion protein cerebral amyloidangiopathy, Progressive subcortical gliosis, Progressive supranuclearpalsy, Subacute sclerosing panencephalitis, and Tangle only dementia.Some of these diseases may also include deposits of fibrillar amyloid βpeptides. For example, Alzheimer's disease exhibits both amyloid βdeposits and tau lesions. Similarly, prion-mediated diseases such asCreutzfeldt-Jakob disease, prion protein cerebral amyloid angiopathy,and Gerstmann-Sträussler-Scheinker syndrome may have also have taulesions. Thus, an indication that a disease is a “tauopathy” should notbe interpreted as excluding the disease from other neurodegenerative ormisfolded and/or aggregated amyloid protein disease classifications orgroupings, which are provided merely as a convenience. The polypeptidesand compositions of the invention may be used to treat neurodegenerativediseases as well as diseases involving tau lesions.

In some embodiments, a polypeptide, pharmaceutical composition, orformulation is for use in a method of reducing amyloid in a patientexhibiting symptoms related to the presence of amyloid or that ispositive for a biomarker associated with a protein misfolding disease,comprising administering to the patient an effective amount of apharmaceutical composition or formulation as described herein. In someembodiments, a polypeptide, pharmaceutical composition, or formulationis for use in a method of maintaining the level of amyloid in a patientexhibiting symptoms related to the presence of amyloid or that ispositive for a biomarker associated with a protein misfolding disease,comprising administering to the patient an effective amount of apharmaceutical composition or formulation as described herein. In someaspects of these embodiments, the biomarker is β-amyloid, which can bedetected with the radiopharmaceutical agent florbetapir (AV-45, EliLilly). In some aspects of these embodiments, the route ofadministration is intrathecal injection or infusion, directintraventricular injection or infusion, intraparenchymal injection orinfusion, or intravenous injection or infusion.

In some embodiments, a polypeptide, pharmaceutical composition, orformulation is for use in a method of disaggregating or remodelingamyloid in a patient. In some embodiments, a polypeptide, pharmaceuticalcomposition, or formulation is for use in a method of reducing amyloidformation in the brain. In some embodiments, a polypeptide,pharmaceutical composition, or formulation of the invention is for usein a method for promoting amyloid clearance in the brain. In someembodiments, a polypeptide, pharmaceutical composition, or formulationof the invention is for use in a method for inhibiting amyloidaggregation in the brain. In some embodiments, a polypeptide,pharmaceutical composition, or formulation of the invention is for usein a method for clearing toxic oligomers in the brain. In someembodiments, a polypeptide, pharmaceutical composition, or formulationof the invention is for use in a method for preventing the formation oftoxic oligomers in the brain. In some embodiments, a polypeptide,pharmaceutical composition, or formulation of the invention is for usein method for protecting neurons from amyloid damage. In someembodiments, a polypeptide, pharmaceutical composition, or formulationis for use in a method of reducing cell-to-cell propagation ofα-synuclein aggregates. In some embodiments, a polypeptide,pharmaceutical composition, or formulation is for use in a method ofblocking cell-to-cell propagation of α-synuclein aggregates. In someaspects of these embodiments, the polypeptide, pharmaceuticalcomposition, or formulation is administered to a patient in need thereofby intrathecal injection or infusion, direct intraventricular injectionor infusion, intraparenchymal injection or infusion, or intravenousinjection or infusion.

In some embodiments, a polypeptide, pharmaceutical composition, orformulation of the invention is for use in a method of causingdisaggregation of Aβ-amyloid deposits in the brain, comprising injectingdirectly into the brain of a patient in need thereof an effective amountof the polypeptide, pharmaceutical composition, or formulation, thuscausing a reduction in Aβ-amyloid deposits in the brain. In otherembodiments, a polypeptide, pharmaceutical composition, or formulationof the invention is for use in a method of causing disaggregation ofAβ-amyloid deposits in the brain, comprising injecting by intravenousdelivery into a patient in need thereof an effective amount of thepolypeptide, pharmaceutical composition, or formulation, thus causing areduction in Aβ-amyloid deposits in the brain.

In one embodiment, a pharmaceutical composition or formulation of theinvention for use in protecting neurons from amyloid damage is givenprophylactically.

In some embodiments, the patient is positive for a biomarker associatedwith a protein misfolding and/or aggregation disease. In one embodiment,the biomarker is β-amyloid and the agent used to detect β-amyloid isflorbetapir (AV45, Eli Lilly).

Unlike prior art antibody-based therapies (e.g., 6E10), GAIM-Ig fusionsas described herein target the core of amyloids rather than unstructuredor partially structured N-terminal residues and demonstrate superiorremodeling activity (FIG. 10A). Thus, in some embodiments, apolypeptide, pharmaceutical composition, or formulation of the inventionis for use in a method of remodeling amyloid. In some aspects of theseembodiments, the route of administration is intrathecal injection orinfusion, direct intraventricular injection or infusion,intraparenchymal injection or infusion, or intravenous injection orinfusion.

In general, the polypeptides disclosed herein bind to amyloid at leastas effectively as M13 phage, g3p, or a variant or fusion protein of g3pdisclosed in the prior art. In some embodiments, the polypeptidesdisclosed herein bind to amyloid more effectively than M13 phage, g3p,or a variant or fusion protein of g3p disclosed in the prior art. Insome embodiments, the polypeptides disclosed herein remodel amyloid moreeffectively than do M13 phage, g3p, or a variant or fusion protein ofg3p disclosed in the prior art. In some embodiments, the polypeptidesdisclosed herein inhibit amyloid aggregation more effectively than doM13 phage, g3p, or a variant or fusion protein of g3p disclosed in theprior art. In some embodiments, the polypeptides disclosed herein cleartoxic oligomers more effectively than do M13 phage, g3p, or a variant orfusion protein of g3p disclosed in the prior art. In some embodiments,the polypeptides disclosed herein reduce cell-to-cell propagation ofα-synuclein aggregates more effectively than do M13 phage, g3p, or avariant or fusion protein of g3p disclosed in the prior art. In someembodiments, the polypeptides disclosed herein detect amyloid moreeffectively than do M13 phage, g3p, or a variant or fusion protein ofg3p disclosed in the prior art. In some embodiments, the polypeptidesdisclosed herein prevent a disease associated with misfolded and/oraggregated amyloid protein more effectively than do M13 phage, g3p, or avariant or fusion protein of g3p disclosed in the prior art. In someembodiments, the polypeptides disclosed herein treat a diseaseassociated with misfolded and/or aggregated amyloid protein moreeffectively than do M13 phage, g3p, or a variant or fusion protein ofg3p disclosed in the prior art. In some embodiments, the polypeptidesdisclosed herein elicit a smaller immune response in a patient ascompared to M13 phage, g3p, or a variant or fusion protein of g3pdisclosed in the prior art. In at least one embodiment, the polypeptidesdisclosed herein do not elicit an immune response in a patient.

In another embodiment, any of the diseases described above may betreated by administration of a nucleic acid molecule of the invention(i.e., encoding a polypeptide comprising a GAIM variant that exhibitsreduced or no immunogenicity and that possesses the ability to bindamyloid, disaggregate/remodel amyloid, and/or inhibit aggregation ofamyloid) alone or associated with a suitable carrier, e.g., a lipidnanoparticle. a polymeric carrier, or a vector, such as a viral vectordirectly to a patient by any suitable route, e.g., inhalation andintravenous infusion. The nucleic acid molecule encoding the polypeptidecomprising a GAIM variant may be DNA or RNA.

Diagnostics

Diagnostic compositions are encompassed by the present invention and maycomprise any of the above-described polypeptides of the invention (e.g.,a polypeptide comprising a GAIM variant, such as a polypeptidecomprising a GAIM-Ig fusion. Thus, in some embodiments, thepolypeptides, pharmaceutical compositions, and formulations describedherein are used in diagnostic applications associated with the variousdiseases described herein. For example, binding of a polypeptide of theinvention to amyloid protein may be used to detect the bound amyloidprotein. Similarly, binding of a polypeptide of the invention when usedas an imaging agent in vivo or in vitro may be part of a diagnosis of aprotein-misfolding, protein-aggregation, or neurodegenerative diseasedescribed herein.

In some embodiments, a polypeptide described herein is used as anamyloid-imaging agent, wherein the imaging agent can detect amyloidprotein and diagnose a disease associated with misfolded and/oraggregated amyloid protein. Because the polypeptides described hereinbind amyloid irrespective of the type of fiber, they can image anddetect any amyloid aggregate (Aβ, tau, α-synuclein, transthyretin,immunoglobulin light chain, etc.) and may diagnose a wide range ofamyloid-associated diseases and conditions. In some embodiments, thepolypeptide used as an amyloid-imaging agent further comprises adetectable label.

Various labels can be attached to a polypeptide comprising a GAIMvariant as described herein using standard techniques for labelingproteins. Examples of labels include fluorescent labels and radiolabels.There are a wide variety of radiolabels that can be used, but in generalthe label is often selected from radiolabels including, but not limitedto, ¹⁸F, ¹¹C, and ¹²³I. These and other radioisotopes can be attached tothe protein using well known chemistry. In one embodiment, the label isdetected using positron emission tomography (PET). However, any othersuitable technique for detection of radioisotopes may also be used todetect the radiotracer.

The polypeptides and compositions of the invention may be used asdiagnostic imaging agents in combination with an imaging agent that isspecific for β-amyloid such as, for example, F18-AV-45, Eli Lilly.Because the use of a diagnostic composition of the invention togetherwith a β-amyloid-specific imaging agent will result in the detection ofnon-β-amyloid aggregates based on differential detection, in oneembodiment, a diagnostic composition of the invention is used as animaging agent in combination with a β-amyloid imaging agent to detectnon-β-amyloid aggregates.

In some embodiments, the polypeptides described herein, or compositionsthereof, are used to detect β-amyloid in the CNS, including the brain.

Diagnostic compositions of the invention may be administered using thesame routes described for therapeutic compositions. In some embodiments,the route of administration is intrathecal injection or infusion, directintraventricular injection or infusion, intraparenchymal injection orinfusion, or intravenous injection or infusion.

Recombinant Techniques

In some aspects, the present invention relates to oligonucleotidescomprising a nucleic acid sequence that encodes a polypeptide of theinvention. For example, the present invention relates to a nucleic acidsequence encoding polypeptides comprising a GAIM variant, includingpolypeptides comprising a GAIM variant attached directly or through asmall linker to an immunoglobulin constant region, as disclosed herein.In general, nucleic acids encoding a polypeptide comprising a GAIMvariant or GAIM-Ig fusion are prepared using conventional recombinantDNA techniques, such as cloning of mutant GAIM domains, direct DNAsynthesis, or by isolating the corresponding DNA from a library using,for example, the M13 sequence as a probe. See, e.g., Sambrook et al.1989, Molecular Cloning A Laboratory Manual, Cold Spring HarborLaboratory, N.Y.; Ausubel et al. 1989, Current Protocols in MolecularBiology, Greene Publishing Associates and Wiley Interscience, N.Y.Nucleic acids encoding a polypeptide comprising a GAIM variant orGAIM-Ig fusion may also be prepared as provided in the Examples below.

For recombinant production, any of the nucleic acid sequences of theinvention may be inserted into an appropriate expression vector whichcontains the necessary elements for the transcription and translation ofthe inserted coding sequence, or in the case of an RNA viral vector, thenecessary elements for replication and translation. The encoding nucleicacid is inserted into the vector in proper reading frame. Accordingly,the invention provides vectors comprising nucleic acids of theinvention. Such vectors include, but are not limited to, DNA vectors,phage vectors, viral vectors, retroviral vectors, etc. Vectors mayinclude, for example, baculovirus, cauliflower mosaic virus, tobaccomosaic virus, Ri plasmid, or Ti plasmid. The choice of appropriatevector in which to clone the nucleic acids of the invention may be madeby those of skill in the art using well-known knowledge of thecompatibility of the vector with the chosen host cell in which to carryout expression. This may be done in any of mammalian cells, plant cells,insect cells, bacterial cells, fungal cells, transgenic animal cells,etc. Exemplary mammalian cells suitable for producing polypeptidesdescribed herein include but are not limited to HEK293 cells,HEK293-derived cells, CHO cells, CHO-derived cells, HeLa cells, and COScells. Exemplary bacterial cells include but are not limited to E. colicells. Exemplary plant cells include but are not limited to duckweedcells. See, e.g., U.S. Pat. No. 8,022,270. Aβ propriate vectors for eachof these cell types are well-known in the art and are generallycommercially available. Nonlimiting exemplary transfection methods aredescribed in Sambrook et al., Molecular Cloning, A Laboratory Manual,3rd ed. Cold Spring Harbor Laboratory Press (2001). Nucleic acids may betransiently or stably transfected in the desired host cells according tomethods known in the art.

In at least one embodiment, the nucleic acid encodes a polypeptidecomprising the amino acid sequence of SEQ ID NO:19. In at least oneembodiment, the nucleic acid encodes a polypeptide comprising the aminoacid sequence of SEQ ID NO:20. In at least one embodiment, the nucleicacid encodes a polypeptide comprising the amino acid sequence of SEQ IDNO:21. In at least one embodiment, the nucleic acid encodes apolypeptide comprising the amino acid sequence of SEQ ID NO:22. In atleast one embodiment, the nucleic acid encodes a polypeptide comprisingthe amino acid sequence of SEQ ID NO:23. In at least one embodiment, thenucleic acid encodes a polypeptide comprising the amino acid sequence ofSEQ ID NO:24. In at least one embodiment, the nucleic acid encodes apolypeptide comprising the amino acid sequence of SEQ ID NO:25. In atleast one embodiment, the nucleic acid encodes a polypeptide comprisingthe amino acid sequence of SEQ ID NO:26.

In at least one embodiment, the nucleic acid encodes a polypeptideconsisting essentially of SEQ ID NO:19 and the Fc portion of a human IgG(e.g., human IgG1). In at least one embodiment, the nucleic acid encodesa polypeptide consisting essentially of SEQ ID NO:20 and the Fc portionof a human IgG (e.g., human IgG1). In at least one embodiment, thenucleic acid encodes a polypeptide consisting essentially of SEQ IDNO:21 and the Fc portion of a human IgG (e.g., human IgG1). In at leastone embodiment, the nucleic acid encodes a polypeptide consistingessentially of SEQ ID NO:22 and the Fc portion of a human IgG (e.g.,human IgG1). In at least one embodiment, the nucleic acid encodes apolypeptide consisting essentially of SEQ ID NO:23 and the Fc portion ofa human IgG (e.g., human IgG1). In at least one embodiment, the nucleicacid encodes a polypeptide consisting essentially of SEQ ID NO:24 andthe Fc portion of a human IgG (e.g., human IgG1). In at least oneembodiment, the nucleic acid encodes a polypeptide consistingessentially of SEQ ID NO:25 and the Fc portion of a human IgG (e.g.,human IgG1). In at least one embodiment, the nucleic acid encodes apolypeptide consisting essentially of SEQ ID NO:26 and the Fc portion ofa human IgG (e.g., human IgG1).

In some embodiments, the nucleic acid encodes a polypeptide consistingessentially of the amino acid sequence of SEQ ID NO:29 (PB108). In someembodiments, the nucleic acid encodes a polypeptide consistingessentially of the amino acid sequence of SEQ ID NO:30 (PB122). In someembodiments, the nucleic acid encodes a polypeptide consistingessentially of the amino acid sequence of SEQ ID NO:31 (PB116). In someembodiments, the nucleic acid encodes a polypeptide consistingessentially of the amino acid sequence of SEQ ID NO:32 (PB114). In someembodiments, the nucleic acid encodes a polypeptide consistingessentially of the amino acid sequence of SEQ ID NO:33 (PB109). In someembodiments, the nucleic acid encodes a polypeptide consistingessentially of the amino acid sequence of SEQ ID NO:34 (PB110). In someembodiments, the nucleic acid encodes a polypeptide consistingessentially of the amino acid sequence of SEQ ID N0:35 (PB105). In someembodiments, the nucleic acid encodes a polypeptide consistingessentially of the amino acid sequence of SEQ ID NO:36 (PB127). Asdescribed above, these embodiments encompass a nucleic acid encoding avariant of SEQ ID NOs: 29, 30, 31, 32, 33, 34, 35, or 36 in which thevariant lacks amino acid 1 (ΔM1), amino acids 1 and 2 (ΔM1 and ΔA2),amino acid 485 (ΔK485), amino acids 1 and 485 (ΔM1 and ΔK485), or aminoacids 1, 2, and 485 (ΔM1, ΔA2, and ΔK485).

In some embodiments, the nucleic acid encoding a polypeptide of theinvention comprises SEQ ID NO:37. In some embodiments, the nucleic acidcomprises SEQ ID NO:38. In some embodiments, the nucleic acid comprisesSEQ ID NO:39. In some embodiments, the nucleic acid comprises SEQ IDNO:40. In some embodiments, the nucleic acid comprises SEQ ID NO:41. Insome embodiments, the nucleic acid comprises SEQ ID NO:42. In someembodiments, the nucleic acid comprises SEQ ID NO:43. In someembodiments, the nucleic acid comprises SEQ ID NO:44. In someembodiments, the nucleic acid comprising any of SEQ ID NOs: 37, 38, 39,40, 41, 42, 43, or 44 further comprises a nucleic acid encoding the Fcportion of IgG (e.g., human IgG1 or IgG2). In at least one embodiment,the nucleic acid encoding an open-stabilized GAIM variant and thenucleic acid encoding the Fc portion of IgG are connected by a nucleicacid encoding a small linker. In at least one embodiment, the nucleicacid encodes the small linker ARS. In some embodiments, the nucleic acidfurther encodes a signal sequence. In some embodiments, the nucleic acidfurther encodes a signal sequence having the 18-amino acid N-terminalsequence of GenBank Ref Seq NP_510891.1.

In at least one embodiment, the nucleic acid encoding a polypeptide ofthe invention is SEQ ID NO:45. In at least one embodiment, the nucleicacid is SEQ ID NO:46. In at least one embodiment, the nucleic acid isSEQ ID NO:47. In at least one embodiment, the nucleic acid is SEQ IDNO:48. In at least one embodiment, the nucleic acid is SEQ ID NO:49. Inat least one embodiment, the nucleic acid is SEQ ID NO:50. In at leastone embodiment, the nucleic acid is SEQ ID NO:51. In at least oneembodiment, the nucleic acid is SEQ ID NO:52.

Vectors used in transformation will usually contain a selectable markerused to identify transformants. In bacterial systems, this can includean antibiotic resistance gene such as ampicillin or kanamycin.Selectable markers for use in cultured mammalian cells include genesthat confer resistance to drugs, such as neomycin, hygromycin, andmethotrexate. The selectable marker may be an amplifiable selectablemarker. One amplifiable selectable marker is the DHFR gene. Anotheramplifiable marker is the DHFRr eDNA (Simonsen and Levinson, PNAS (1983)80:2495). Selectable markers are reviewed by Thilly (Mammalian CellTechnology, Butterworth Publishers, Stoneham, Mass.) and the choice ofselectable markers is well within the level of ordinary skill in theart. The expression elements of the expression systems vary in theirstrength and specificities. Depending on the host/vector systemutilized, any of many suitable transcription and translation elements,including constitutive and inducible promoters, may be used in theexpression vector. For example, when cloning in bacterial systems,inducible promoters such as pL of bacteriophage λ, plac, ptrp, ptac(ptrp-lac hybrid promoter), and the like may be used; when cloning ininsect cell systems, promoters such as the baculovirus polyhedronpromoter may be used; when cloning in plant ceil systems, promotersderived from the genome of plant cells (e.g., heat shock promoters; thepromoter for the small subunit of RUBISCO; the promoter for thechlorophyll a/b binding protein) or from plant viruses (e.g., the 35SRNA promoter of CaMV; the coat protein promoter of TMV) may be used;when cloning in mammalian cell systems, promoters derived from thegenome of mammalian cells (e.g., metallothionein promoter) or frommammalian viruses (e.g., the adenovirus late promoter; the vacciniavirus 7.5 K promoter) may be used; when generating cell lines thatcontain multiple copies of expression product, SV40-, BPV- and EBV-basedvectors may be used with an appropriate selectable marker. In caseswhere plant expression vectors are used, the expression of sequencesencoding linear or non-cyclized forms of the expression product of theinvention may be driven by any of a number of promoters. For example,viral promoters such as the 35S RNA and 19S RNA promoters of CaMV(Brisson et al., Nature (1984) 31 0:511-514), or the coat proteinpromoter of TMV (Takamatsu et al., EMBO J (1987) 6:307-311) may be used;alternatively, plant promoters such as the small subunit of RUBISCO(Coruzzi et al., EMBO J. (1984) 3:1671-1680; Broglie et al., Science(1984) 224:838-843) or heat shock promoters, e.g., soybean hsp17.5-E orhsp17.3-B (Gurley et al., Mol. Cell. Biol. (1986) 6:559-565) may beused. These constructs can be introduced into plant cells using Tiplasmids, Ri plasm ids, plant virus vectors, direct DNA transformation,microinjection, electroporation, etc. See, e.g., Weissbach & Weissbach1988, Methods for Plant Molecular Biology, Academic Press, NY, SectionVIII, pp. 421-463; and Grierson & Corey 1988, Plant Molecular Biology,2d Ed., Blackie, London, Ch. 7-9. In one insect expression system thatmay be used to produce proteins of the invention, Autographa californicanuclear polyhidrosis virus (AcNPV) is used as a vector to express theforeign genes. The virus grows in Spodoptera frugiperda cells. A codingsequence may be cloned into non-essential regions (for example, thepolyhedron gene) of the virus and placed under control of an AcNPVpromoter (for example, the polyhedron promoter). Successful insertion ofa coding sequence results in inactivation of the polyhedron gene andproduction of non-occluded recombinant virus, i.e. virus lacking theproteinaceous coat coded for by the polyhedron gene. These recombinantviruses are used to infect Spodoptera frugiperda cells in which theinserted gene is expressed. See, e.g., Smith et al., J. Viral. (1983)46:584; U.S. Pat. No. 4,215,051. Further examples of this expressionsystem may be found in Ausubel et al., eds. 1989, Current Protocols inMolecular Biology, Vol. 2, Greene Publish. Assoc. & Wiley Interscience.

In mammalian host cells, any of several viral based expression systemsmay be used. In cases where an adenovirus is used as an expressionvector, a coding sequence may be ligated to an adenovirustranscription/translation control complex, e.g., the late promoter andtripartite leader sequence. This fusion gene may then be inserted in theadenovirus genome by in vitro or in vivo recombination. Insertion in anon-essential region of the viral genome (e.g., region E1 or E3) willresult in a recombinant virus that is viable and capable of expressingpeptide in infected hosts (see, e.g., Logan & Shenk, PNAS (1984)81:3655). Alternatively, the vaccinia 7.5 K promoter may be used (see,e.g., Mackett et al., PNAS (1982) 79:7415; Mackett et al., J. Viral.(1984) 49:857; Panicali et al., PNAS (1982) 79:4927). Other viralexpression systems include adeno-associated virus and lentiviruses.

In another embodiment, the invention provides a host cell harboring thevector containing a nucleic acid of the invention. Methods oftransfecting or transforming or otherwise getting a vector of theinvention into a host cell are known in the art. A cell harboring thevector, when cultured under appropriate conditions, will produce thepolypeptides of the invention. As noted above, suitable host cellsinclude but are not limited to mammalian cells, transgenic animal cells,plant cells, insect cells, bacterial cells, and fungal cells. Forexample, suitable host cells include but are not limited to HEK293cells, HEK293-derived cells, CHO cells, CHO-derived cells, HeLa cells,and COS cells.

Host cells comprising nucleic acid constructs (e.g., vectors) are grownin an appropriate growth medium. As used herein, the term “appropriategrowth medium” means a medium having nutrients required for the growthof cells. The recombinantly-produced polypeptides of the invention canbe isolated from the culture media using techniques known in the art.

Specific examples of vectors and cells used for the recombinantproduction of the polypeptides of the invention are set forth in theExamples below.

EXAMPLES Example 1: TauK18P301L Expression, Purification and FiberAssembly

Human TauK18P301L fragment corresponding to residues 244-372 of Tau-441(2N4R) with the P213L mutation were expressed and purified as describedfor tau-MTBR (Krishnan et al. (2014) J Mol Biol, 426:2500-19).Tau-K18P301L fibers were assembled by adding 40 μM low-molecular weightheparin (Fisher Scientific) to 40 μM TauK18P301L monomer in 0.1 M sodiumacetate pH 7.0 buffer containing 2 mM DTT and incubating for 3 days at37° C. Fiber formation was confirmed by Thioflavin T (ThT).

Example 2: Aβ Fiber Assembly

Aβ1-42 (rPeptide), N-truncated Aβ11-42 (Bachem), Aβ11-42-Pyro (AnaSpec),Aβ3-42-Pyro (AnaSpec) and Aβ1-42-E22Q (AnaSpec) were dissolved inhexafluoroisopropanol (HFIP) and incubated at room temperature for 24hours until a clear solution developed. The peptide solution was driedunder vacuum for 1 h. Fibers were assembled as described by Stine etal., 2003. One hundred micrograms Aβ peptide was dissolved in 40 μLDMSO, diluted to 1140 μL in 10 mM HCl solution, and incubated withshaking at 500 rpm for 24 hours at 37° C. Fiber formation was confirmedby ThT.

Example 3: Generation of GAIM-Ig Fusion Proteins

Site-specific mutagenesis of the control scaffold PB120 was performed inβ-strands facing the inner groove of the GAIM domains (FIG. 1A). Theseβ-strands, 4 and 5 in the N1 domain and 9 and 10 in the N2 domain,facilitate inter-domain interactions in the closed state of GAIM andprevent the exposure of the ToIA binding site (Hoffman-Thoms et al.(2013) J Biol Chem, 288:12979-91) previously shown to in part overlapwith the amyloid binding motif in GAIM (Krishnan et al. (2014) J MolBiol, 426:2500-19). In addition, sites in the N2-hinge region involvedin N1-N2 domain assembly and specific regions in N2 important for F-pilibinding were mutated (Weininger et al. (2009) PNAS, 106:12335-40; Dengand Perham (2002) J Mol Biol, 319:603-14) to investigate how GAIMamyloid binding activity translates to its function during phageinfection.

GAIM-Ig fusion proteins were expressed using the Expi293™ ExpressionSystem (Thermo Fisher Scientific) according to manufacturer'sinstructions. Purification of the proteins was performed on HiTrap®MabSelect™ SuRe™ column (GE Healthcare Lifesciences) in 20 mM sodiumphosphate, pH 7.0 followed by a gradient elution in 20 mM sodium acetatefrom pH 4.0 to pH 3.6 over 20 CV using ΔKTA™ Pure FPLC system. Fusionproteins were dialyzed into D-PBS pH 7 and filter sterilized(Ultrafree®-MC spin columns, Millipore). Protein purity was analyzed byNuPAGE™ 4-12% Bis-Tris gel system with MES SDS Running Buffer (ThermoFisher Scientific) followed by InstantBlue™ Staining Solution(Expedeon). In addition, analytical SEC was used to assess the purity ofGAIM IgG-fusions using a TSKgel® G3000SW XL, 7.8 mm ID×30 cm, 5 μMcolumn (TOSOH BIOSCIENCES) in an UltiMate™ 3000 UHPLC focused system(ThermoFisher Scientific). For each sample, 7.5 μg of protein wasinjected onto the SEC column, separation was performed in D-PBS mobilephase at a flow rate of 0.5 mL/min. Peak purity was analyzed usingChromeleon™ 7 software. GAIM IgG-fusion variants were synthesized byATUM.

Example 4: Generation of GAIM Dimers

GAIM dimers were generated from the GAIM-Ig fusions using theFabRICATOR® (IdeS) enzyme (Genovis), which specifically clips the fusionat the immunoglobulin hinge to yield GAIM dimers linked by two disulfidebonds (FIG. 1C), for 2 hours at 37° C. Cleavage was followed byseparation from Fc by Capto™ Adhere according to the manufacturer'sprotocol. Purity of GAIM dimer was confirmed on a NuPAGE™ 4-12% Bis-Trisgel system separated in MES SDS Running Buffer. GAIM dimer (0.5 μM) wasincubated for 2 hours at 25° C. in 100 mM potassium phosphate, pH 7.0with increasing concentrations of Guanidine (Sigma). The fluorescencewas measured in 10-mm cells at 310 nm and 340 nm after excitation at 280nm and at 360 nm after excitation at 295 nm. The data were analyzedusing a two-state folding model assuming a linear dependence offluorescence emissions on guanidine hydrochloride concentration. Afterconfirming expected molecular size and clearance of Fc fragments onSDS-PAGE gel, the protein was subjected to unfolding studies.

Example 5: Thermal Unfolding of GAIM Monitored by SYPRO® Orange BindingAssay

A SYPRO® Orange binding assay was carried out to monitor GAIM domainseparation and stability of the N2 domain. SYPRO® Orange binds poorly tothe closed conformation of folded GAIM in an aqueous solution. When thetwo domains of GAIM dissociate and expose hydrophobic residues, the dyebinds to the exposed hydrophobic surfaces and shows increasedfluorescence. One micromolar GAIM-Ig fusion in PBS was mixed with ×20excess of SYPRO® Orange (Invitrogen cat. no. S-6650) in a 96-well plateand sealed. Thermal unfolding was monitored in a Roche LightCycler® 480RT-PCR by continuous increase in temperature from 20° C. to 95° C. at arate of 0.24° C./minute. Excitation was set to 465 nm and emission at580 nm with melt factor at 1, quant factor at 10 and maximum integrationtime for two seconds. The arbitrary unit of fluorescent signal wasrecorded and normalized to a scale of 0-100 (Layton and Hellinga, 2011).

Thermal unfolding of the GAIM monomer monitored by SYPRO® Orange bindingshowed a single transition around 43° C. that corresponds to the domainopening and N2 unfolding transition (FIGS. 4A-4B). GAIM dimers obtainedaccording to Example 4 showed identical melting profiles to monomers insolution, whereas GAIM-Ig fusions in solution showed three distincttransitions upon thermal unfolding (FIGS. 4A-4B). The first transition,Tm1, occurred around 44° C., as seen in the GAIM-monomers and dimers(FIG. 4B). Two additional transitions at 64° C. and 81° C. are like theFc domains unfolding transitions (Traxlmayr et al., 2012, BiochimBiophys Acta, 1824:524-529). Comparing the GAIM-specific Tm1 showed thatthe GAIM and Fc domains remain as independent folding domains and no newstructural elements are generated in the chimeric molecule.

Example 6: GAIM Retains Its Native Conformational Stability in the IgGFusion Dimer

The conformational stability of GAIM in the IgG-fusion was investigatedusing guanidine hydrochloride (GuHCI)-induced unfolding of GAIM dimer byintrinsic fluorescence. GAIM dimers were generated as described inExample 4 and were equilibrated in 0, 2, and 5 M GuHCI solutions at 25°C. for 2 hours. Selective excitation of the Trp residues at 295 nmshowed a minimal change in GAIM dimer fluorescence intensities between 0and 2 M concentrations, with no change in the emission Amax (345 nm)(FIG. 5A). At 5 M GuHCI, the Trp fluorescence was red-shifted by 15 nm(Amax 360 nm) and the fluorescence intensity was significantly higherthan both 0 and 2 M samples. GAIM dimers were then excited at 280 nm(Trp and Tyr residues) and the fluorescence emission spectra wasrecorded (FIG. 5B). The fluorescence emission intensity at 340 nmdecreased between 0 and 2 M GuHCI and then increased by a similar marginat 5 M GuHCI. Similar spectral changes were also observed in g3p (Martinand Schmid, 2003, J Mol Biol, 328:863-75), indicating that GAIM in theGAIM-Ig fusion variants retained the native conformational stability ofGAIM in native g3p.

We generated detailed denaturation profiles of GAIM dimers by recordingfluorescence emission intensities at 310, 340, and 360 nm in a range ofconcentrations of GuHCI. GAIM dimers showed a biphasic denaturationprofile at 340 nm when excited at 280 nm (FIG. 5C). The first transitionoccurred between 1 and 2 M GuHCI and the next between 2 and 3 M GuHCI.We then fitted the 310 nm (excitation 280 nm) and 360 nm (excitation 295nm) denaturation profiles to a two-state protein unfolding model (FIGS.5D-5E) and calculated the N2 and N1 domain denaturation transitions at1.5 M and 2.6 M GuHCI respectively. The first transition at 1.5 M GuHCIrepresents the separation of the two domains N1 and N2 and thesimultaneous unfolding of the less stable N2 domain. The secondtransition represents the unfolding of the more stable N1 domain at 2.6M GuHCI. These values correspond to previously-reported denaturationtransitions in g3p (Martin and Schmid, 2003, J Mol Biol, 328:863-75).Therefore, these data indicate that each GAIM in the GAIM dimer in theGAIM-Ig fusion forms an independent folding unit and adopts aconformation similar to the g3p tip protein from filamentous phages.

Example 7: GAIM-Ig Fusions Bind Aβ and Tau Fibers

Fifty microliters of Aβ fibers (0.8 μM) or tauK18P301L fibers (1 μM) in50 mM carbonate buffer, pH 9.6 was added per well in a 96-well MaxiSorp®plate (Thermo Fisher) and incubated 16 hours at 4° C. Wells were washed3 times with DPBS-Tween (0.05%) and 2 times with DPBS, followed byblocking with SuperBlock™ (Thermo Scientific) for 1.5 hours at roomtemperature. Wells were washed 3 times with PBS. GAIM-Ig fusion wasadded in high concentration PBS-T (14.7 mM KH₂PO₄, 80.6 mM Na₂HPO₄-7H₂O,27 mM KCl, 1.38 M NaCl, 0.05% tween) at the indicated concentrations andincubated at 37° C. for 2 hours followed by 3 washes in DPBS-Tween(0.05%) and 3 washes with DPBS. Human specific Fc-HRP antibody (JacksonImmunoResearch, cat. no. 109-035-008) diluted 1:5000 in DPBS-Tween(0.05%) containing 0.2% gelatin was added for 45 minutes at 37° C. After2 washes in DPBS-Tween (0.05%) and 2 washes in DPBS the signal wasdeveloped with TMB solution (Thermo Fisher), the reaction was stopped bythe addition of 0.25 N HCl and the absorbance at 450 nm was recordedwith a Tecan Infinite® M1000 PRO plate reader.

Most of the mutations in N1 and N2 residues facing the inner groove ofGAIM were found to affect the in Aβ1-42 fiber binding by ELISA (FIG.6A). Binding activities of the mutated GAIM variants ranged from 0.7 nMto 175 nM EC₅₀, representing a more than 250-fold change in bindingaffinity to fAβ42. There was a strong correlation (P-value 10⁻⁴,r_(s)=0.703) between binding efficacy (EC₅₀) and the first meltingtransition (Tm1). A decrease in Tm1 represents a more open conformationof GAIM with increased binding, stabilized variants with higher Tm1 tendto lose their binding activity. This is suggestive of an amyloid fiberbinding motif in GAIM being exposed when the inter-domain interactionsare weakened and agrees with previous data showing that GAIM binding istemperature dependent (Krishnan et al. (2014) J Mol Biol, 426:2500-19).

To discern whether the change in binding activity for fAβ42 translatesto other amyloids proteins, a subset of the variants was tested by ELISAfor binding to amyloid fibers formed from the microtubule-binding regionof tau. Comparing GAIM-Ig variants' binding activities (EC₅₀) for fAβ42and ftau (P-value 10⁻⁴, r_(s)=0.878; FIG. 6B) showed a tight correlationin the binding activity of GAIM-Ig (P-value 10⁻⁴, r_(s)=0.862) for thetwo different amyloids.

Example 8: Superior Binding of Open-Stabilized GAIM-Ig Fusion ProteinsOver Stabilized GAIM-Ig Fusion Proteins

Several mutations in GAIM that stabilized the N2 domain and favored astronger interaction with the N1 domain led to decreased amyloidbinding. Elimination of a proline containing loop in the N2 domain bysubstituting the Q₁₅₆GTDPVK₁₆₂ loop (SEQ ID NO:7) with QGGK (SEQ IDNO:10) increased Tm1 by 3.6° C. and resulted in 18-fold loss of fAβ42binding (FIG. 7A). Likewise, amino acid substitutions of F₁₃₅QNN₁₃₈ (SEQID NO:5) to FQGN (SEQ ID NO:8) and R₁₄₃QGA₁₄₆ (SEQ ID NO:6) to VNGV (SEQID NO:9) stabilized N2 (Tm1) by 1.8° C. and 2.5° C. respectively. Thesestabilized variants showed reduced fAβ42 binding activity compared tothe GAIM scaffold. (FIG. 7A). Similarly, introducing the Q128H mutation,which stabilizes the interactions of the N2 hinge subdomain and N1,decreased fiber binding (FIG. 7A). The substitutions of T1, T2, or T3 inthe N2 domain all reduced non-specific binding to collagen, although theQ128H showed a marginal 1.4-fold increase (FIG. 9). Such mutantsindicate an inverse relationship between the potency of a GAIM variant'samyloid-binding activity and its stability.

To create a more open conformation of GAIM, the D24DKTLD29 (SEQ ID NO:3)loop in N1 (FIG. 3A) was replaced with the homologous sequence EGDS (SEQID NO:4) from the filamentous phage IF1 and tested in combination withN2-stabilizing mutations (e.g., at one or more turns/loops indicated inFIG. 3C). The presumed open and N2-stabilized GAIM variants were testedfor protein quality and amyloid-binding activity. All open-stabilizedvariants display improved fiber-binding activity with EC₅₀<1.5 nM;consistent with a more exposed and accessible amyloid fiber-binding site(FIG. 7B). One exception was the EGDS (SEQ ID NO: 4) variant with asuper stabilized N2 (PB113; SEQ ID NO:17; Tm1=52.7° C.), containing allthree N2-stabilizing mutations combined (F₁₃₅QGN₁₃₈, V₁₄₃NGV₁₄₆, andQ₁₅₆GGK₁₆₂) (respectively, SEQ ID NOs:8, 9, 10), resulting in a loss offAβ42 binding activity (FIG. 7B). This could be due to major structuralchanges in N2 masking the amyloid interaction site(s) in GAIM, either byintroducing intra-domain interactions or by over-stabilizing the N2domain. The open-stabilized variants with increased fiber binding lostthe correlation between Tm1 and fAβ42 binding, suggesting an uncouplingof amyloid binding site(s) and N2-stability in these variants. AllEGDS-N2 (“EGDS” disclosed as SEQ ID NO: 4) stabilized variants displayedgood protein quality by SDS-PAGE and presented as monomers by sizeexclusion chromatography (SEC). Additionally, there was a shift inretention time by SEC that further indicated a more open GAIM conformermolecule.

Example 9: GAIM-Ig Fusions Target Multiple Amyloids with DiverseMorphologies

Open-stabilized GAIM-Ig fusions were tested for the ability to engagedifferent types and conformations of Aβ aggregates. Various modified Aβpeptides were fibrillized and binding affinities of GAIM-Ig fusions tothese aggregates were measured. N-truncated Aβ11-42, Aβ11-42-Pyro,Aβ3-42-Pyro and Aβ1-42-E22Q-Dutch mutation (Levy et al, 1990; VanBroeckhoven et al, 1990) were aggregated under the same conditions asAβ42 and fiber formation was verified by ThT (FIGS. 8A-8D) and by TEM(data not shown). The aggregates formed using these peptides showed verydiverse morphologies. For example, pyro-glu 3-42 forms fibers that haveseveral bends in their structure, E22Q variant forms smooth long fibersand 11-42 peptides form several short fibers. Both the open-stabilizedvariant PB108 and the scaffold PB120 were found to engage these fibersby ELISA. PB108 showed ˜20-fold improved binding to the variousaggregates compared to PB120, EC₅₀ 0.9-1.9 nM (FIGS. 8A-8D). Similarlysuperior binding was observed for the other tested open-stabilizedGAIM-Ig fusions (Table 2).

TABLE 2 Open-Stabilized GAIM-Ig Fusions Bind and Remodel AmyloidProtein. GAIM-Ig fAβ42 fTauKL fAβ42 Fusion Binding Binding RemodelingProtein (nM) (nM) (%) PB108 1 15 87 PB122 1.3 24 80 PB116 0.8 7.0 82PB114 0.9 8.7 86 PB109 0.8 5.2 82 PB110 0.9 7.6 92 PB105 0.8 ND* NDPB127 0.8 ND ND *ND = no data collected

TABLE 3 Open-Stabilized GAIM-Ig Fusions Targets a Variety of AmyloidProteins. PB120 PB108 Binding Target EC₅₀ (nM) EC₅₀ (nM) Aβ(1-42) 18.00.8 (0.5 nM K_(D) by SPR) Aβ(11-42) 24.3 0.9 Aβ3-42 PyroE3 24.1 1.1Aβ11-42 PyroE11 40.6 1.9 Aβ1-42 E22Q 21.9 1.0 TauKL 59 14 Wild-type TTR105 7 LC lambda_1 (variable + ND* 24 constant) LC lambda_1 (variable) ND1.3 LC lambda_2 (variable) ND 19 *ND = no data collected. Bindingdetermined by ELISA unless otherwise indicated.

The ability of open-stabilized GAIM-Ig fusions to bind different typesand conformations of amyloid protein is further shown in Tables 2 and 3.For example, both Tables 2 and 3 show binding of open-stabilized GAIM-Igfusions to Aβ42 and to tauKL with low nanomolar affinity, and Table 3further demonstrates their binding to morphologically-diverseimmunoglobulin light chain (LC) and transthyretin (TTR) aggregates withlow nanomolar affinity. These data show superior targeting across adiverse array of amyloid fibers as compared to the control scaffold andare consistent with previous NMR studies showing that GAIM engages themid and C-terminal sequences in Aβ42 fibers (Krishnan et al. (2014) JMol Biol, 426:2500-19).

Example 10: GAIM-Ig Fusions Bind Amyloid Protein Specifically

To rule out non-specific binding, GAIM-Ig fusions were tested at highconcentrations (1.8 μM, 100-fold higher than the EC₅₀ for a truesubstrate like fAβ42) for off-target binding to other fibrillar specieslike collagen. Two hundred twenty-five nanograms per well of humancollagen (Sigma cat. no. C5483) in D-PBS was immobilized on MaxiSorp®96-well plates (Thermo Fisher Scientific) for 16 hours at 37° C.followed by blocking in SuperBlock™ (Thermo Fisher Scientific) for 1hour at room temperature. GAIM-Ig fusion in PBS-Tween (0.05%) wasincubated at 37° C. for 1 hour followed by 3×5-minute washes inPBS-Tween (0.05%). Human specific Fc-HRP antibody (JacksonImmunoResearch, cat. no. 109-035-008) was added 1:5000 in PBS-Tween(0.05%) for 45 minutes at 37° C. followed by 3×5-minute washes inPBS-Tween (0.05%) and 2×5-minute washes in PBS. The signal was developedwith TMB solution (Sigma), the reaction was stopped by the addition of0.25 N HCl and the absorbance at 450 nm was recorded with a TecanInfinite® M1000 PRO plate reader. GAIM-Ig fusions showed minimal bindingto non-amyloid substrates by ELISA (Krishnan et al. (2014) J Mol Biol,426:2500-19).

Example 11: Open-Stabilized GAIM-Ig Fusions Exhibit Enhanced Remodelingof Aβ Fibers

Remodeling assays were carried out in low retention microfuge tubes(Fisher Scientific 02-681-320). Buffers used in these assays contain0.05% sodium azide to prevent microbial growth. To make sure there wasno protease contamination in any sample, all remodeled complexes wererun on an SDS-PAGE gel and checked for any degradation. For assays using<1 μM fibers, protein quality was instead confirmed using western blotanalysis.

Aβ42 fibers (2.5 μM) were co-incubated with or without GAIM-Ig fusionvariants for three days at 37° C. Aliquots of the complexes were thenincubated with varying concentrations of urea. The ThT fluorescence ofthe complexes in urea was plotted against the urea concentration.Remodeling efficiency at a fixed urea concentration was plotted aspercent loss in ThT fluorescence compared to fibers without any GAIM-Igfusion treatment.

GAIM-Ig fusions with different Aβ42 fiber binding potencies wereselected to determine if the remodeling efficiencies depend on thebinding potencies and open conformational state. FIG. 10A showsremodeling efficiencies of different GAIM fusions incubated with Aβ42fibers under identical conditions and concentrations. Open-stabilizedvariants with low nanomolar fAβ42 binding showed a 2-fold to 3-foldincrease in remodeling activity, with an average remodeling activity of83% compared to the control scaffold (35%). FIG. 10B reveals a positivecorrelation between altered fAβ42-binding and remodeling activity, suchthat GAIM-Ig fusions with superior Aβ binding also exhibit superiorremodeling activity.

FIGS. 10A-10C, as well as transmission electron microscopy data (seeExample 13; FIG. 10D), further demonstrate that open-stabilized GAIM-Igfusions remodel amyloid fibers to cause a loss of fibrillararchitecture, as opposed to merely sticking to and masking fibrillarstructures. For example, when incubated in increasing concentrations ofurea but without exposure to a GAIM-Ig fusion, Aβ42 fibers resisteddenaturation and showed less than 10% structural change in 1 M urea asmeasured by ThT fluorescence (FIG. 10C). In higher urea concentrations,ThT fluorescence dropped dramatically, suggesting loss of fibrillarstructure. In contrast, fibers treated with sub-stoichiometric amountsof GAIM-Ig fusion began to show 30-90% reduced ThT binding at 1 M urea.This finding suggests that the GAIM-Ig fusions bind and alter thefibrillar structures to a state that cannot bind ThT and that GAIMremodeling activity varies between different GAIM Ig-fusions, withopen-stabilized GAIM-Ig fusions demonstrating high remodeling activity.

Example 12: Open-stabilized GAIM-Ig Fusions Exhibit Enhanced Remodelingof TauK18P301L Fibers

Remodeling assays were also performed by co-incubating Tau-K18P301Lfibers with GAIM-Ig fusions to demonstrate that remodeling of aggregatesis generic to amyloid protein.

Remodeling assays were carried out in low retention microfuge tubes(Fisher Scientific 02-681-320). Buffers used in these assays contain0.05% sodium azide to prevent microbial growth. To make sure there wasno protease contamination in any sample, all remodeled complexes wererun on an SDS-PAGE gel and checked for any degradation. For assays using<1 μM fibers, protein quality was instead confirmed using western blotanalysis.

Unlike fAβ42 fibers, Tau-k18P301L fibers readily dissolve in lowconcentration urea solutions. Thus, the remodeling efficiencies ofGAIM-Ig fusions against Tau-K18P301L was investigated using sarkosylsolubility assays. TauK18P301L fibers (1 μM) were co-incubated with orwithout GAIM-Ig-fusion variants at 37° C. for 5 days. Fibers and thecomplexes were incubated with or without 1% sarkosyl for 15 minutes andspun down at 100,000 g for 30 minutes. The supernatant from each samplewas carefully removed and loaded on a 4-12% NuPAGE® gels (Invitrogen).Proteins were transferred to a Nitrocellulose membrane and probed forTauK18P301L. The percent remodeling was calculated by quantifying thegel bands using a Biorad Chemidoc™ system.

Fibers assembled in vitro using Tau-K18P310L (not exposed to a GAIM-Igfusion) showed resistance to dissolution when incubated with 1 sarkosyl.GAIM-Ig fusion-treated Tau-K18P301L fibers dissolved in 1% sarkosyl morereadily than untreated fibers (FIG. 11A), indicating that these fiberscan also be remodeled like fAβ42. When incubated with varyingconcentrations of GAIM-Ig fusions, these fibers dissolved in aconcentration-dependent manner (FIG. 11B).

The remodeling efficiency of PB120 were compared with the remodelingefficiencies of open-stabilized GAIM IgG fusions. PB113, asuper-stabilized, disulfide-free GAIM with reduced infectivity (Katheret al., 2005, J Mol Biol, 354:666-78) has no binding activity to Aβ1-42or Tau-K18P301L fibers (data not shown) and was added as a negativecontrol. The open-stabilized GAIM-Ig fusion proteins showed enhancedremodeling activities as compared to PB120 (FIG. 11B), whereas PB113 hadno remodeling activity (FIG. 11A).

We investigated whether GAIM fusions remodel Tau-K18P301L fibers andrelease soluble TauK18P301L species when we co-incubate the samples. AtGAIM fusion concentrations yielding potent remodeling (e.g., 10-250 nM),there were no soluble species seen in supernatant of complexes that havenot been subjected to sarkosyl treatment (FIG. 11A), suggesting that theremodeled material does not liberate soluble TauK18P301L species ormonomers upon binding GAIM fusions.

Example 13: Open-Stabilized GAIM-Ig Fusions Cause Amyloid Fibers to LoseFibrillar Architecture

Aβ1-42 fibers (15 μl of 20 μM sample) were applied on carbon coatedcopper grids (TedPella cat #01844-F). Samples were then washed gentlywith 0.5 ml water, inverted and floated over a drop of 2% uranyl acetatesolution. After 30 seconds, the grids were removed and dried by wickingout the excess liquid from the edge of the grids using a filter paper.FEI Tecnai™ Spirit TEM was used to image the fibers. FIG. 10D shows arepresentative TEM images of Aβ42 fibers incubated withsub-stoichiometric open-stabilized GAIM-Ig fusion. When exposed toopen-stabilize GAIM-Ig fusions, Aβ42 fibers lost their fibrillararchitecture (FIG. 10D). Similarly, TEM analysis showed thatTau-K18P301L fibers lost their characteristic fibrillar conformationwhen co-incubated with an open-stabilized GAIM-Ig fusion (FIG. 11C).

Example 14: Open-Stabilized GAIM-Ig Fusions Exhibit Increased Inhibitionof Amyloid Aggregation

Open-stabilized GAIM-Ig fusion proteins were tested for assemblyinhibition activity by co-incubation with Aβ42 monomers at 37° C. for 10hours. Amyloid fiber formation was followed by ThT fluorescence andcompared to fiber formation without GAIM as well as in the presence ofthe negative control PB113.

One hundred micrograms of HFIP-treated Aβ1-42 (rPeptide) monomericsample was dissolved in 80 μl DMSO, mixed thoroughly by pipetting,vortexed, and diluted in 5.4 mL D-PBS to a final Aβ1-42 concentration of4.04 μM. GAIM-Ig fusion samples were diluted in PBS to intermediatestock solutions of 10, 2.5, 0.63, and 0.16 μM. Eighty microliters Aβ1-42monomer solution was distributed in each well of a black, round bottom96-well plate (LVL, cat. no. 225.LS.PP). Ten microliters of each GAIM-Igfusion stock solution were added to wells containing Aβ1-42, followed bythe addition of 10 μl ThT (33 μM in PBS), for a final concentration of3.2 μM Aβ1-42 and 3.3 μM ThT per well. The plate was sealed withtransparent film and ThT fluorescence at 430/485 nm (Ex/Em) was recordedevery 20 minutes for 14 hours in a Tecan Infinite® M1000 PRO platereader while incubated at 37° C. with 3-second vertical shaking every 20minutes. The percentage of Aβ42 aggregation for each GAIM-Ig fusionconcentration was calculated relative to non-treated Aβ42 wells(positive control wells).

Open-stabilized GAIM-Ig fusions showed dose-dependent assemblyinhibition when added for 10 hours at the indicated concentrations (FIG.12A). The open-stabilized fusions further showed increased assemblyinhibition activity compared to the control scaffold, PB120 (FIGS.12A-12B). For example, at 250 nM, representative open-stabilized GAIM-Igfusions PB108 and PB116 showed a 40-20% increase in blocking fiberformation compared to PB120 (FIG. 12B). The ability ofsub-stoichiometric amounts of open-stabilized GAIM-Ig fusions topotently block amyloid fibril formation suggests that the fusions blockamyloid fiber formation by binding to core β strands in the growingfiber or seeds involved in nucleation dependent fiber assembly (Krishnanet al. (2014) J Mol Biol, 426:2500-19).

Similarly, open-stabilized GAIM-Ig fusions showed dose-dependentassembly inhibition against tau fibers (FIG. 12C). TauK18P301L assemblyreactions were set by incubating 10 μM tau monomers in 0.1 M sodiumacetate pH 7.0 buffer with 2 μM low-molecular weight heparin (FisherScientific) at 37° C. for 3 days in the presence of varyingconcentrations of GAIM fusions (0-500 nM). ThT fluorescence of theassembly reactions were recorded by diluting samples to 1 μM into a 5 μMThT solution. Inhibitory effects of GAIM on TauK18P301L assembly werecalculated by comparing the assembly of TauK18P301L without GAIM fusion.

In vitro fiber assembly of full-length tau or truncated sequences suchas MTBR or K18 requires the presence of heparin to promote nucleationand subsequent assembly. The tested GAIM fusions inhibited ftauKLassembly in the presence of heparin. Moreover, the open-stablized GAIMfusions blocked nucleation three-fold to five-fold better as compared toPB120 (FIG. 12C-12D). Together, these results indicate thatopen-stabilized GAIM fusions bind both Aβ and tau on-pathwayintermediates and inhibit assembly of amyloid aggregates.

1. A polypeptide comprising a variant of starting amino acid sequenceSEQ ID NO:16, wherein the variant differs from SEQ ID NO:16 by one ormore sets of amino acid changes selected from: a) substitution of T50with any amino acid and H55T; b) N137G; c) N142A; d) R143V and Q144N; orR143V, Q144N, and A146V; or R143V, Q144N, and A146T; or R143V, Q144N,and A146K; and e) Q156V, G157N, ΔT158, ΔD159, ΔP160, and V161G; orQ156Y, G157N, ΔT158, ΔD159, ΔP160, and V161G; or G157N, ΔT158, ΔD159,ΔP160, and V161G; or ΔT158, ΔD159, ΔP160, and V161G; wherein the variantoptionally further differs from SEQ ID NO:16 by one or more sets ofamino acid changes selected from: f) ΔM1; or ΔM1 and ΔA2; and g)substitution of N38 with any amino acid other than cysteine; orsubstitution of N38 with any amino acid other than cysteine, andsubstitution of G40 with any amino acid other than cysteine; orsubstitution of G40 with any amino acid other than cysteine, threonine,or serine.
 2. The polypeptide of claim 1, wherein the T50 substitutionis selected from the group consisting of T50G, T50H, T50K, and T50R. 3.The polypeptide of claim 2, wherein the T50 substitution is T50H.
 4. Thepolypeptide of claim 1, wherein the variant differs from SEQ ID NO:16 atleast by ΔM1 and ΔA2.
 5. The polypeptide of any one of claims 1-4,wherein the variant differs from SEQ ID NO:16 at least by N137G and/orN142A.
 6. The polypeptide of claim 4, wherein the variant furtherdiffers from SEQ ID NO:16 by one or more sets of amino acid changesselected from: a) substitution of T50 with any amino acid and H55T; andb) R143V and Q144N; or R143V, Q144N, and A146V; or R143V, Q144N, andA146T; or R143V, Q144N, and A146K;
 7. The polypeptide of claim 6,wherein the T50 substitution is selected from the group consisting ofT50G, T50H, T50K, and T50R.
 8. The polypeptide of claim 7, wherein theT50 substitution is T50H.
 9. The polypeptide of claim 1, wherein thevariant of SEQ ID NO:16 is selected from the group consisting of SEQ IDNO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ IDNO:24, SEQ ID NO:25, and SEQ ID NO:26.
 10. The polypeptide of any one ofclaims 1-9, further comprising an immunoglobulin constant region. 11.The polypeptide of claim 10, wherein the immunoglobulin constant regionsequence is the Fc portion of a human IgG.
 12. The polypeptide of claim11, consisting essentially of the polypeptide of any one of claims 1-9and the Fc portion of a human IgG.
 13. The polypeptide of claim 12,wherein the human IgG is human IgG1.
 14. A polypeptide consistingessentially of an amino acid sequence selected from: a) SEQ ID NO:29; b)SEQ ID NO:30; c) SEQ ID NO:31; d) SEQ ID NO:32; e) SEQ ID NO:33; f) SEQID NO:34; g) SEQ ID NO:35; and h) SEQ ID NO:36; wherein the variantoptionally has one or more sets of amino acid changes selected from: i)ΔM1; or ΔM1 and ΔA2; and j) ΔK485.
 15. A pharmaceutical compositioncomprising the polypeptide of any one of claims 1-14 and apharmaceutically acceptable carrier.
 16. A method of reducing amyloid,inhibiting amyloid formation, inhibiting amyloid aggregation, orremoving and/or preventing the formation of toxic oligomers in a subjectin need thereof, comprising administering to the subject a polypeptideof any one of claims 1-14 or the pharmaceutical composition of claim 15.17. The method of claim 16, wherein the amyloid or oligomers comprise aprotein selected from Androgen receptor; apolipoprotein AI;apolipoprotein AII; apolipoprotein AIV; aposerum amyloid A; Aβ; ABri;ADan; Atrophin-1; atrial natriuretic factor; ataxin; calcitonin;γ-crystallin; cystatin C; fibrinogen; gelsolin; huntingtin; insulin;islet amyloid polypeptide; immunoglobulin kappa light chain;immunoglobulin lambda light chain; kerato-epithelin; keratin;lactahedrin; lactoferrin; lysozyme; lung surfactant protein C; medin;odontogenic ameloblast-associated protein; prion protein; procalcitonin;prolactin; semenogelin I; serum amyloid A; superoxide dismutase I;β2-microglobulin; TATA box binding protein; tau; transthyretin; andα-synuclein.
 18. A method of treating a disease in a subject in needthereof, wherein the disease is selected from Alzheimer's disease; earlyonset Alzheimer's disease; late onset Alzheimer's disease;presymptomatic Alzheimer's disease; AL amyloidosis; amyotrophic lateralsclerosis (ALS); Amyotrophic lateral sclerosis/parkinsonism-dementiacomplex; Argyrophilic grain dementia; Aortic medial amyloidosis; ApoAIamyloidosis; ApoAII amyloidosis; ApoAIV amyloidosis; Atrial amyloidosis;British/Danish dementia; Cataract; Corticobasal degeneration; Cornealamyloidosis associated with trichiasis; cystatin C plaque-relateddisease; cystatin C plaque-related coronary disease; cystatin Cplaque-related kidney disease; cutaneous lichen amyloidosis; Dementiapugilistica; dentatorubral-pallidoluysian atrophy; diffuseneurofibrillary tangles with calcification; dementia with Lewy bodies;Down's syndrome; Familial Amyloidotic Cardiomyopathy (FAC); FamilialAmyloidotic Polyneuropathy (FAP); Familial British dementia; FamiliaDanish dementia; familial encephalopathy; Familial Mediterranean fever;Fibrinogen amyloidosis; Finnish hereditary amyloidosis; Frontotemporaldementia with Parkinsonism; frontotemporal lobar degeneration (FTLDs);frontotemporal lobe dementia; Hallervorden-Spatz disease;Hemodialysis-related amyloidosis; hereditary cerebral amyloidangiopathy; hereditary cerebral hemorrhage with amyloidosis; hereditarylattice corneal dystrophy; Huntington's disease; Icelandic hereditarycerebral amyloid angiopathy; Inclusion-body myositis;Injection-localized amyloidosis; islet amyloid polypeptide amyloidosis;Lysozyme amyloidosis; multiple myeloma; Myotonic dystrophy; Niemann-Pickdisease type C; Non-Guamanian motor neuron disease with neurofibrillarytangles; Parkinson's disease; peripheral amyloidosis; Pick's disease;Pituitary prolactinoma; Postencephalitic parkinsonism; Prion proteincerebral amyloid angiopathy; prion-mediated disease; kuru;Creutzfeldt-Jakob disease (CJD); Gerstmann-Sträussler-Scheinker disease(GSS); fatal familial insomnia (FFI); scrapie; spongiformencephalopathy; pulmonary alveolar proteinosis; Progressive subcorticalgliosis; Progressive supranuclear palsy; Senile Systemic Amyloidosis;serum AA amyloidosis; spinal and bulbar muscular atrophy;spinocerebellar ataxia (SCA1, SCA3, SCA6, or SCA7); Subacute sclerosingpanencephalitis; systemic amyloidosis; familial amyloidosis; wild-typeamyloidosis; Tangle only dementia; and Tauopathies, comprisingadministering to the subject a polypeptide of any one of claims 1-14 orthe pharmaceutical composition of claim
 15. 19. The method of claim 18,wherein the disease is selected from Parkinson's disease, Alzheimer'sdisease, and Huntington's disease.
 20. The method of claim 19, whereinthe disease is Alzheimer's disease.
 21. The method of claim 18, whereinthe disease is a prion-mediated disease selected from Creutzfeldt-Jakobdisease, kuru, fatal familial insomnia, andGerstmann-Sträussler-Scheinker disease.
 22. An oligonucleotidecomprising a nucleic acid encoding a polypeptide of any one of claims1-14.
 23. The oligonucleotide of claim 22, wherein the nucleic acidencoding the polypeptide comprises a sequence selected from SEQ IDNO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ IDNO:42, SEQ ID NO:43, and SEQ ID NO:44.
 24. The oligonucleotide of claim22, wherein the nucleic acid encoding the polypeptide consists of asequence selected from SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ IDNO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, and SEQ ID NO:52.
 25. Avector comprising the nucleic acid of any one of claims 22-24.
 26. Ahost cell comprising the vector of claim
 25. 27. The host cell of claim26, wherein the host cell is selected from the group consisting of aninsect cell, a fungal cell, a plant cell, a bacterial cell, a mammaliancell, and a transgenic animal cell.
 28. The host cell of claim 27,wherein the host cell is selected from the group consisting of HEK293cells, HEK293-derived cells, CHO cells, CHO-derived cells, HeLa cells,and COS cells.
 29. A method of making a protein that binds amyloid,comprising expressing the protein encoded by the nucleic acid in thevector of claim 25 and isolating the expressed protein.
 30. A proteinthat binds amyloid, produced by expressing the protein encoded by thenucleic acid in the vector of claim 25 and isolating the expressedprotein.
 31. The protein of claim 30, wherein the protein is expressedby culturing the host cell of any one of claims 26-28.
 32. Use of apolypeptide of any one of claims 1-14 or the pharmaceutical compositionof claim 15 in the manufacture of a medicament for reducing amyloid,inhibiting amyloid formation, inhibiting amyloid aggregation, orremoving and/or preventing the formation of toxic oligomers in a subjectin need thereof.
 33. The use of claim 32, wherein the amyloid oroligomers comprise a protein selected from Androgen receptor;apolipoprotein AI; apolipoprotein AII; apolipoprotein AIV; aposerumamyloid A; Aβ; ABri; ΔDan; Atrophin-1; atrial natriuretic factor;ataxin; calcitonin; γ-crystallin; cystatin C; fibrinogen; gelsolin;huntingtin; insulin; islet amyloid polypeptide; immunoglobulin kappalight chain; immunoglobulin lambda light chain; kerato-epithelin;keratin; lactahedrin; lactoferrin; lysozyme; lung surfactant protein C;medin; odontogenic ameloblast-associated protein; prion protein;procalcitonin; prolactin; semenogelin I; serum amyloid A; superoxidedismutase I; β2-microglobulin; TATA box binding protein; tau;transthyretin; and α-synuclein.
 34. Use of a polypeptide of any one ofclaims 1-14 or the pharmaceutical composition of claim 15 in themanufacture of a medicament for treating a disease selected fromAlzheimer's disease; early onset Alzheimer's disease; late onsetAlzheimer's disease; presymptomatic Alzheimer's disease; AL amyloidosis;amyotrophic lateral sclerosis (ALS); Amyotrophic lateralsclerosis/parkinsonism-dementia complex; Argyrophilic grain dementia;Aortic medial amyloidosis; ApoAI amyloidosis; ApoAII amyloidosis; ApoAIVamyloidosis; Atrial amyloidosis; British/Danish dementia; Cataract;Corticobasal degeneration; Corneal amyloidosis associated withtrichiasis; cystatin C plaque-related disease; cystatin C plaque-relatedcoronary disease; cystatin C plaque-related kidney disease; cutaneouslichen amyloidosis; Dementia pugilistica; dentatorubral-pallidoluysianatrophy; diffuse neurofibrillary tangles with calcification; dementiawith Lewy bodies; Down's syndrome; Familial Amyloidotic Cardiomyopathy(FAC); Familial Amyloidotic Polyneuropathy (FAP); Familial Britishdementia; Familia Danish dementia; familial encephalopathy; FamilialMediterranean fever; Fibrinogen amyloidosis; Finnish hereditaryamyloidosis; Frontotemporal dementia with Parkinsonism; frontotemporallobar degeneration (FTLDs); frontotemporal lobe dementia;Hallervorden-Spatz disease; Hemodialysis-related amyloidosis; hereditarycerebral amyloid angiopathy; hereditary cerebral hemorrhage withamyloidosis; hereditary lattice corneal dystrophy; Huntington's disease;Icelandic hereditary cerebral amyloid angiopathy; Inclusion-bodymyositis; Injection-localized amyloidosis; islet amyloid polypeptideamyloidosis; Lysozyme amyloidosis; multiple myeloma; Myotonic dystrophy;Niemann-Pick disease type C; Non-Guamanian motor neuron disease withneurofibrillary tangles; Parkinson's disease; peripheral amyloidosis;Pick's disease; Pituitary prolactinoma; Postencephalitic parkinsonism;Prion protein cerebral amyloid angiopathy; prion-mediated disease; kuru;Creutzfeldt-Jakob disease (CJD); Gerstmann-Sträussler-Scheinker disease(GSS); fatal familial insomnia (FFI); scrapie; spongiformencephalopathy; pulmonary alveolar proteinosis; Progressive subcorticalgliosis; Progressive supranuclear palsy; Senile Systemic Amyloidosis;serum AA amyloidosis; spinal and bulbar muscular atrophy;spinocerebellar ataxia (SCA1, SCA3, SCA6, or SCA7); Subacute sclerosingpanencephalitis; systemic amyloidosis; familial amyloidosis; wild-typeamyloidosis; Tangle only dementia; and Tauopathies.
 35. The use of claim34, wherein the disease is selected from Parkinson's disease,Alzheimer's disease, and Huntington's disease.
 36. The use of claim 35,wherein the disease is Alzheimer's disease.
 37. The use of claim 34,wherein the disease is a prion-mediated disease selected fromCreutzfeldt-Jakob disease, kuru, fatal familial insomnia, andGerstmann-Sträussler-Scheinker disease.
 38. A polypeptide of any one ofclaims 1-14 or the pharmaceutical composition of claim 15 for use inreducing amyloid, inhibiting amyloid formation, inhibiting amyloidaggregation, or removing and/or preventing the formation of toxicoligomers in a subject in need thereof.
 39. The polypeptide orpharmaceutical composition for use of claim 38, wherein the amyloid oroligomers comprise a protein selected from Androgen receptor;apolipoprotein AI; apolipoprotein AII; apolipoprotein AIV; aposerumamyloid A; Aβ; ABri; ΔDan; Atrophin-1; atrial natriuretic factor;ataxin; calcitonin; γ-crystallin; cystatin C; fibrinogen; gelsolin;huntingtin; insulin; islet amyloid polypeptide; immunoglobulin kappalight chain; immunoglobulin lambda light chain; kerato-epithelin;keratin; lactahedrin; lactoferrin; lysozyme; lung surfactant protein C;medin; odontogenic ameloblast-associated protein; prion protein;procalcitonin; prolactin; semenogelin I; serum amyloid A; superoxidedismutase I; β2-microglobulin; TATA box binding protein; tau;transthyretin; and α-synuclein.
 40. A polypeptide of any one of claims1-14 or the pharmaceutical composition of claim 15 for use in treating adisease in a subject in need thereof, wherein the disease is selectedfrom Alzheimer's disease; early onset Alzheimer's disease; late onsetAlzheimer's disease; presymptomatic Alzheimer's disease; AL amyloidosis;amyotrophic lateral sclerosis (ALS); Amyotrophic lateralsclerosis/parkinsonism-dementia complex; Argyrophilic grain dementia;Aortic medial amyloidosis; ApoAI amyloidosis; ApoAII amyloidosis; ApoAIVamyloidosis; Atrial amyloidosis; British/Danish dementia; Cataract;Corticobasal degeneration; Corneal amyloidosis associated withtrichiasis; cystatin C plaque-related disease; cystatin C plaque-relatedcoronary disease; cystatin C plaque-related kidney disease; cutaneouslichen amyloidosis; Dementia pugilistica; dentatorubral-pallidoluysianatrophy; diffuse neurofibrillary tangles with calcification; dementiawith Lewy bodies; Down's syndrome; Familial Amyloidotic Cardiomyopathy(FAC); Familial Amyloidotic Polyneuropathy (FAP); Familial Britishdementia; Familia Danish dementia; familial encephalopathy; FamilialMediterranean fever; Fibrinogen amyloidosis; Finnish hereditaryamyloidosis; Frontotemporal dementia with Parkinsonism; frontotemporallobar degeneration (FTLDs); frontotemporal lobe dementia;Hallervorden-Spatz disease; Hemodialysis-related amyloidosis; hereditarycerebral amyloid angiopathy; hereditary cerebral hemorrhage withamyloidosis; hereditary lattice corneal dystrophy; Huntington's disease;Icelandic hereditary cerebral amyloid angiopathy; Inclusion-bodymyositis; Injection-localized amyloidosis; islet amyloid polypeptideamyloidosis; Lysozyme amyloidosis; multiple myeloma; Myotonic dystrophy;Niemann-Pick disease type C; Non-Guamanian motor neuron disease withneurofibrillary tangles; Parkinson's disease; peripheral amyloidosis;Pick's disease; Pituitary prolactinoma; Postencephalitic parkinsonism;Prion protein cerebral amyloid angiopathy; prion-mediated disease; kuru;Creutzfeldt-Jakob disease (CJD); Gerstmann-Sträussler-Scheinker disease(GSS); fatal familial insomnia (FFI); scrapie; spongiformencephalopathy; pulmonary alveolar proteinosis; Progressive subcorticalgliosis; Progressive supranuclear palsy; Senile Systemic Amyloidosis;serum AA amyloidosis; spinal and bulbar muscular atrophy;spinocerebellar ataxia (SCA1, SCA3, SCA6, or SCA7); Subacute sclerosingpanencephalitis; systemic amyloidosis; familial amyloidosis; wild-typeamyloidosis; Tangle only dementia; and Tauopathies.
 41. The polypeptideor pharmaceutical composition for use of claim 40, wherein the diseaseis selected from Parkinson's disease, Alzheimer's disease, andHuntington's disease.
 42. The polypeptide or pharmaceutical compositionfor use of claim 41, wherein the disease is Alzheimer's disease.
 43. Thepolypeptide or pharmaceutical composition for use of claim 40, whereinthe disease is a prion-mediated disease selected from Creutzfeldt-Jakobdisease, kuru, fatal familial insomnia, andGerstmann-Sträussler-Scheinker disease.